CN113226360A - Methods, systems, and compositions for novel uses of enterobactin to treat iron deficiency and related anemia - Google Patents

Abstract

In one embodiment, the present invention relates to the use of enterobactin (Ent) and/or an Ent analogue as a therapeutic agent for the treatment of iron deficiency and iron deficiency related anemia. In preferred embodiments, Ent and/or the Ent analog can be delivered to a host organism, such as a human subject, to treat an iron-related disease condition. In such embodiments, the Ent and/or the Ent analog can be delivered to a human subject in need thereof by a pharmaceutical composition and/or by a genetically engineered bacterium or probiotic organism.

Description

Methods, systems, and compositions for novel uses of enterobactin to treat iron deficiency and related anemia

Cross Reference to Related Applications

This international PCT application claims the benefit and priority of U.S. provisional application No. 62/700,480 filed on 19.7.2018. The entire specification and drawings of the above-referenced application are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to novel benefits of individual microbiota-derived molecules in host animals. For example, enterobactin (Ent) secreted by bacteria is an iron-scavenging siderophore with putative negative effects on the host. However, the high incidence of Ent-producing commensals in the human gut indicates a potential host mechanism for the beneficial use of Ent for the treatment of disease conditions associated with iron metabolism and specific metro deficiency. Through novel and unique assays, the inventors of the present invention discovered an unexpected and surprising role for Ent in supporting growth and labile iron pools in exemplary eukaryotic host organisms. The inventors of the present invention demonstrated that Ent promotes mitochondrial iron uptake and surprisingly does so by binding to the ATP synthase alpha subunit, which functions intragranularly independently of ATP synthase. The inventors of the present invention have also demonstrated that this mechanism is retained in mammalian cells. This study reveals a new paradigm of "iron tug-of-war" between commensals and their hosts and important mechanisms for mitochondrial iron uptake and homeostasis.

Background

Based on analysis by the World Health Organization (WHO) and other literature (Stevens et al, 2013; WHO,2015), iron deficiency is the most common nutritional deficiency condition and the most common cause of anemia, affecting a global population beyond 1/4, particularly women and children. Anemia is also the cause of disability in about 9% of the world. Importantly, oral iron supplementation, the primary treatment for this condition, presents serious problems. First, the efficacy of oral iron supplementation is very low, in part because oral iron supplementation induces hormonal changes that block iron uptake (increased hepcidin) (Muckenthaler et al, 20l7) (Cook and Reddy, 1995; Moretti et al, 2015). Secondly, this treatment has well-known adverse side effects that may lead to increased mortality, especially in people with other diseases (Sazawal et al, 2006). For example, oral iron supplementation is known to promote inflammation by inducing free radicals and adverse changes in the composition of the human microbiota that may be responsible for some of the adverse side effects (Jaeggi et al, 2015; Kortman et al, 2015; Lund et al, 1999; Tang et al, 2017). It is also known that anemia is very common in people with common GI diseases such as Inflammatory Bowel Disease (IBD) (more than 50% of IBD patients in the united states also have anemia (koutrouubakis et al, 2015)). Many anemic patients, including those with other GI diseases, are completely intolerant of oral iron supplementation, requiring intravenous infusion that is also well known to be associated with health risks and side effects (Auerbach and Macdougall, 2017; Munoz et al, 2009). Defects in the iron transport system are the cause of certain iron-deficiency anemias (Brissot et al, 2011) and increased iron uptake efficiency may be critical for revolutionary therapy for most anemic patients.

As detailed below, the inventors of the present invention demonstrate that enterobactin (Ent) has the potential to increase iron absorption efficiency without high levels of oral iron supplementation and its associated side effects. In fact, this finding was unexpectedly clinically significant and was recognized by experts in their review in top-grade journals (Anderson, 2018). For example, an article in New England medical Journal (New England Journal of Medicine) (NEJM) at 11.2018 highlighted The novel and unexpected clinical significance of The inventors' underlying research and highlighted The prediction of The ability of Ent to move Iron into mitochondria in several cell types in humans (Gregory Anderson: "Iron competition-Host counterattack (Iron Wars-The Host Strikes Back)" NEJM 2018) (FIG. 17). Notably, Ent is a catecholase siderophore produced almost exclusively by enterobacteria to clear iron from the environment. Given that siderophores are known to be key virulence mediators of pathogens, the clearance of Ent is expected to negatively impact host iron homeostasis and certain cellular processes. To inhibit the growth of pathogenic bacteria that rely on Ent to scavenge iron from host cells, the mammalian immune system produces the Ent binding protein, lipoprotein 2, which sequesters Ent. This defense system may negatively impact host iron pools and animal function. More importantly, this mechanism does not explain how the host animal will deal with a large amount of Ent from the non-infectious gut microbiota, of which enterobacter is the most prevalent commensal microorganism in humans and caenorhabditis elegans (c. Given the symbiotic relationship between commensal e.coli (e.coli) and the host, there may be unknown beneficial mechanisms that have evolved in animals to use bacterial Ent for host iron homeostasis.

Iron transport into mitochondria is a key event in iron homeostasis, as many poorly cell-labile irons are transported into mitochondria for incorporation into heme and Fe-S complexes (Muckenthaler et al, 2017). Under normal conditions, about 70% of the iron in humans is present in hemoglobin (Zhang and ens, 2009). Since the iron-binding step of heme biosynthesis occurs in mitochondria, transport of iron into mitochondria is critical for hemoglobin production or erythropoiesis. In anemic conditions with low hemoglobin counts, a higher proportion of iron needs to be transported into the mitochondria.

In fact, the use of supplemented Ent by bacteria may have several beneficial effects. In one embodiment, this may involve having more Ent likely to increase the incidence of Ent-utilizing bacteria (primarily e.coli in humans), which may have a positive therapeutic effect in some cases. In fact, too little commensal E.coli may cause anemia or other unhealthy conditions. The incidence of commensal E.coli varies between populations. In another example, it is now well known that changes in the composition of the gut microbiota (towards unhealthy aspects) are very significant negative side effects of taking oral iron supplements. If taking Ent allows for a drastic reduction in the effective iron supplement dosage, patients are more likely to receive a strong overall benefit by potentially minimizing adverse changes in the composition of the gut microbiota.

In addition, as part of the immune response in a mammalian host, expression of lipocalin 2 is induced by pathogen attack and it is known that lipocalin 2 binds to Ent in order to sequester Ent and its bound iron from benefit to the proliferation of certain infectious bacteria. Thus, adding more Ent under this infection condition may interfere with the role of lipocalin 2in combating these bacteria. However, this problem may not be a serious impediment to the potential therapeutic use of Ent for the treatment of anemia. First, because the anti-infective effect of lipoprotein 2 does create a low iron environment for the bacteria, oral supplementation with high levels of iron would likely have a greater effect on iron-deficient infectious bacteria than it would have if Ent compromised the program by lipoprotein 2in anemic patients. It is to be remembered that bacteria can take up iron in a way that is independent of Ent, and that bacteria only require Ent under low iron conditions. Thus, potential side effects of Ent in this regard have been present in anemic patients who need to take oral iron.

Thus, there remains a substantial need in the art to identify and characterize the molecular components and host interactions involved in bacterial Ent molecular biology.

In particular, there is a need for new systems, methods and compositions for developing Ent-based therapies and pharmaceutical compositions that can be used to modulate and treat iron-related disease conditions in animals and humans.

Disclosure of Invention

In one embodiment, the inventors of the present invention have demonstrated a unique and sensitive assay for testing the effect of e. In particular, the inventors of the present invention have identified a new paradigm for the effect of symbiotic produced siderophores (enterobacterin or Ent) on host physiology. The inventors of the present invention found that Ent promotes mitochondrial iron levels in host animals and beneficially affects host development. Ent performs this function by binding to the host mitochondrial ATP synthase alpha subunit, and this binding is independent of the entire ATP synthase complex. This previously unknown mechanism can counteract bacterial Ent clearance and its effect on the host labile iron pool (figure 7), and this function should enhance the symbiotic relationship between microorganisms and animals. By retaining this function between C.elegans and humans, the present invention can be consistent with a high incidence of enterobacteria in the gut of both animal species and the ability of commensal E.coli in mammals to produce Ent. This novel inventive technique presents a new paradigm for competition between microorganisms and host cells (iron "tug of war"), which is distinct from the well-studied function of mammalian lipoprotein 2in binding to Ent as a defense mechanism against pathogenic bacteria (fig. 7).

Iron deficiency is one of the most common nutritional disorders threatening the health of a large number of children and women in the world (WHO, 2002). The composition and behavior of the human gut microbiome that produces various iron-binding siderophores may have a great impact on the development and treatment of this condition. The present invention demonstrates that Ent and Ent supplementation in animal and human models promote iron uptake and growth under both low iron and high iron conditions, which in turn can indicate that the levels of iron in the gut are typically not high enough to result in complete inhibition of Ent production from the gut microbiota. The profound effect of additional Ent supplementation on iron levels and animal growth under iron deficiency conditions (fig. 2G) suggests that disruption of microbial composition may significantly contribute to human iron deficiency disorders and act as a surrogate for iron deficiency

One embodiment of the present invention may significantly contribute to the potential of Ent supplementation as a treatment for this widespread human health problem.

The inventors of the present invention have also provided experimental evidence: ATP synthase alpha subunit is unlikely to promote mitochondrial iron uptake in a co-transport model, where end-Fe may simply ride up when ATP synthase alpha subunit is transported into the mitochondria. In contrast, in one embodiment of the present invention, a transport retention model is presented in which the ATP synthase α subunit within the mitochondria binds and retains end-Fe, which means that end-Fe can enter and leave the mitochondria through passive mechanisms or systems involving protein transporters. Recent studies in mammalian cells have shown that Ent can enter mammalian cells by infiltration, while studies in yeast have indicated transporters that can transport Ent into fungal cells. In one example, passive diffusion appears to be more direct in the retention model, as Ent-Fe will also leave the mitochondria without interacting with the ATP synthase alpha subunit.

The present technology further demonstrates that the role of the ATP synthase alpha subunit in mitochondrial iron retention may not be as good as ATP synthaseClosing; it requires neither interaction with other subunits nor its enzymatic activity. Thus, Ent-Fe³⁺May be capable of interacting with an ATP synthase alpha subunit located within mitochondria, but physically separated from ATP synthase. However, in one example, it is possible that the α subunit is located away from the site of ATP synthase in wild-type animals, and thus, even though Ent may be able to interact with the α subunit in the absence of the β subunit, Ent may still bind to the α subunit that is associated with ATP synthase under normal conditions.

Iron uptake into mitochondria contributes to a large extent to the regulation of unstable iron pool levels, but the mechanism of this process remains to be understood. In the analysis of C.elegans by the present inventors, the effect of Ent and ATP-1 on unstable iron levels was very profound (FIGS. 2 and 4). These effects suggest that this newly discovered system involving Ent and ATP synthase alpha subunits represents an important mechanism behind iron uptake into mitochondria, thereby understanding that Ent-producing enterobacteriaceae are the most prevalent commensal microorganisms in both caenorhabditis elegans and humans. In addition, certain embodiments of the inventive techniques described herein may indicate mechanisms that may have a significant impact on understanding other systems involved in iron transport into mitochondria. For example, the retention model may also be involved in the function of other siderophores, including mammalian siderophores. The present technology described herein further demonstrates the value of caenorhabditis elegans to study the effects of metabolites produced by individual microbiota on host physiology and symbiotic relationships between animals and gut microbes and provides therapeutic Ent supplementation of humans and animals that may have or are at risk for iron deficiency-related disease conditions.

Thus, the present invention identifies and characterizes bacterial Ent as a major regulator and characterization of animal iron levels and growth. One object of the present invention can comprise systems, methods and compositions for novel assays in exemplary eukaryotic model organisms, in this case caenorhabditis elegans, which elucidates the role of bacterial Ent in supporting animal growth and one or more iron levels.

Another aspect of the invention comprises systems, methods, and compositions demonstrating the interaction between Ent and the ATP synthase alpha subunit that promotes mitochondrial iron uptake in both caenorhabditis elegans and mammals.

Yet another aspect of the invention comprises the use of Ent to promote host iron homeostasis via its interaction with the ATP synthase alpha subunit. In this preferred embodiment, Ent supplementation may be utilized as a therapeutic to treat one or more iron deficiency-related disease conditions. In this preferred embodiment, a therapeutically effective amount of end supplementation may be introduced to a subject in need thereof. Delivery of Ent may be by pharmaceutical composition or even by introduction of genetically engineered probiotics and/or symbionts configured to produce/overproduce exogenous, modified and/or endogenous Ent.

Another aspect of the invention may further comprise systems, methods and compositions for treating iron deficiency-related conditions and their associated anemia. In a preferred embodiment, such systems, methods and compositions for treating and/or prophylactically preventing iron deficiency-related conditions and anemia related thereto may comprise end supplementation as described herein.

Additional aspects of the invention may include systems, methods, and compositions for using Ent and/or ATP-1 as biomarkers of iron-related disease conditions and diagnostic biomarkers for diagnosing iron deficiency-related disease conditions and/or susceptibility of a subject to iron deficiency-related disease conditions.

Additional aspects of the invention may include one or more of the following embodiments:

1. a method of treating iron deficiency in a subject in need thereof, comprising administering a therapeutically effective amount of enterobacterin (Ent) or a pharmaceutically acceptable salt thereof.

2. The method of embodiment 1, wherein the therapeutically effective amount of Ent is isolated.

3. The method of embodiment 1, wherein said therapeutically effective amount of Ent comprises an analogue of Ent wherein said therapeutically effective amount of Ent.

4. The method of embodiment 3, wherein the Ent analog is selected from the group consisting of: TRENCAM, SERSAM, SER (3M) SAM, TRENSAM and TREN (3M) SAM.

5. The method of embodiment 1 or 4, wherein said Ent or said Ent analog is combined with a pharmaceutically acceptable carrier.

6. The method of embodiment 5, wherein the pharmaceutically acceptable carrier is a nutritional supplement.

7. The method of embodiment 1, wherein the iron deficiency comprises iron deficiency anemia.

8. The method of embodiments 1 and 7, wherein the subject in need thereof comprises a human subject.

9. A method of preventing iron deficiency in a subject in need thereof, comprising administering a prophylactically effective amount of enterobacterin (Ent), or a pharmaceutically acceptable salt thereof.

10. The method of embodiment 9, wherein the therapeutically effective amount of Ent is isolated.

11. The method of embodiment 9, wherein said therapeutically effective amount of Ent comprises an Ent analog wherein said therapeutically effective amount of Ent.

12. The method of embodiment 11, wherein the Ent analog is selected from the group consisting of: TRENCAM, SERSAM, SER (3M) SAM, TRENSAM and TREN (3M) SAM.

13. The method of embodiment 9 or 14, wherein said Ent or said Ent analog is combined with a pharmaceutically acceptable carrier.

14. The method of embodiment 13, wherein the pharmaceutically acceptable carrier is a nutritional supplement.

15. The method of embodiment 9, wherein the iron deficiency comprises iron deficiency anemia.

16. The method of embodiments 9 and 15, wherein the subject in need thereof comprises a human subject.

17. A therapeutic agent for treating iron deficiency in a subject, the therapeutic agent comprising an active ingredient represented by the following general formula (I):

and a pharmaceutically acceptable carrier.

18. The method of embodiment 17, wherein the therapeutically effective amount of the compound of formula I is isolated.

19. The method of embodiment 17, wherein the therapeutically effective amount of the compound of formula I comprises an analog thereof.

20. The method of embodiment 19, wherein the analog of the compound of formula I is selected from the group consisting of:

21. the method of embodiment 17 or 20, wherein the compound of formulae I-VI is combined with a pharmaceutically acceptable carrier.

22. The method of embodiment 21, wherein the pharmaceutically acceptable carrier is a nutritional supplement.

24. The method of embodiment 17, wherein the iron deficiency comprises iron deficiency anemia.

25. The method of embodiments 17 and 24, wherein the subject in need thereof comprises a human subject.

26. The method of embodiments 6,14 and 22, wherein the nutritional supplement comprises a probiotic.

27. A genetically modified probiotic for treating iron deficiency in a subject in need thereof, the genetically modified probiotic comprising a probiotic configured to express a heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of enterobacterin (Ent).

28. The genetically modified bacterium of embodiment 27, wherein the probiotic comprises an enterobacter probiotic.

29. The genetically modified bacterium of embodiment 27, wherein the heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of Ent comprises a heterologous nucleotide operably linked to a promoter encoding one or more genes selected from the group consisting of: entA, entB, entC, entD, entE and entF.

31. The genetically modified bacterium of embodiment 27, wherein the heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of Ent comprises a heterologous nucleotide operably linked to a promoter encoding one or more of the amino acid sequences selected from the group consisting of: SEQ ID NO. 1-6.

31. The genetically modified bacterium of embodiment 27, wherein the subject in need thereof is a human subject.

32. The genetically modified bacterium of embodiment 31, wherein the iron deficiency comprises iron deficiency anemia.

33. A nutraceutical composition for treating iron deficiency in a subject in need thereof, comprising a probiotic configured to express a heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of enterobacterin (Ent) and an excipient.

34. The nutraceutical composition of embodiment 33, wherein the probiotic comprises an enterobacter probiotic.

35. The nutraceutical composition of embodiment 33, wherein the heterologous nucleotides operably linked to a promoter encoding one or more genes for the biosynthesis of Ent comprise heterologous nucleotides operably linked to a promoter encoding one or more genes selected from the group consisting of: entA, entB, entC, entD, entE and entF.

36. The nutraceutical composition of embodiment 33, wherein the heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of Ent comprises a heterologous nucleotide operably linked to a promoter encoding one or more of the amino acid sequences selected from the group consisting of: SEQ ID NO. 1-6.

37. The nutraceutical composition of embodiment 33, wherein the subject in need thereof is a human subject.

38. The nutraceutical composition of embodiment 37, wherein the iron deficiency comprises iron deficiency anemia.

This application is directed to various journal articles and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the examples and claims.

Drawings

The above and other aspects, features and advantages of the present disclosure will be better understood from the following detailed description taken in conjunction with the accompanying drawings, which are given by way of illustration only and do not limit embodiments of the present disclosure, and in which:

FIG. 1: the microbial metabolite enterobactin (Ent) supports caenorhabditis elegans development. (A) Sketches, microscope images and bars showing that trace amounts of live bacteria support post-embryonic growth of worms fed on heat-inactivated e. The volume of worms was measured 4 days after the larvae were placed on the plates. (B-C) bacterial mutant screening identified 5 genes in the Enterobacteriaceae (Ent) biosynthetic pathway that supported host development when each of these 5 mutants was fed under assay conditions (as indicated by a reduction in worm volume). In the screening (B), the enzyme (C) was identified as being red in color. (D) The growth defects caused by feeding live E.coli mutants of either entA or entF as well as heat inactivated E.coli were completely suppressed by dietary supplementation with Ent. A representative microscope image is shown in fig. 8A. (E) Supplementation with 2,3-DHBA rescued the growth of worms feeding the entera mutant bacteria, but not the entF mutant bacteria, confirming that only the end product Ent was beneficial for worm growth. (F) Growth defects caused by feeding of Ent live E.coli mutants together with heat inactivated E.coli were not phenotypically mimicked by mutation of the gene fepA encoding E.coli Ent iron receptor, suggesting that Ent cannot benefit worm growth by its bacterial iron scavenging effect. See also fig. 8B and 8C for the effect of fepA and worm images. (G) Supplementation with other siderophores (fluorescein or ferrichrome) did not rescue the growth of worms fed with the entF mutants along with heat-inactivated food. Toxicity testing of these siderophores is shown in FIGS. 8F-H. (H) Results of whole worm lysate CAS staining showing that the end levels of worms fed with entF mutant bacteria were significantly lower than the end levels of worms fed with wild type bacteria. (I) The sketches, bar graphs and statistical analysis of the feeding conditions showed that worm larvae fed only with live entera or entF e.coli strains (lawn) showed reduced growth rates compared to worms fed with parental wild-type e.coli, indicating that even though Ent is not absolutely required under this feeding condition, host development significantly benefited from Ent. For all figures, "n" is the number of worms assessed. Data are presented as mean ± SEM. P <0.00 l. All data represent at least three independent experiments.

FIG. 2: bacterial enterobacterin promotes host iron pool levels. (A-E) feeding condition sketches, fluorescence images and bar graphs depicting the effect of feeding conditions on host iron levels and pftn-2:: GFP expression. (A) Helminths fed either entera or entF e.coli with heat-inactivated e.coli showed a large increase in calcein-AM fluorescence (indicating a decrease in unstable iron levels) and this change was completely suppressed by dietary supplementation of Ent. (B) Iron reactivity reporter gene pftn-2 expression of GFP was reduced in worms fed either entA or entF E.coli and heat-inactivated E.coli. (C and D) Ent supplementation of heat-inactivated E.coli restored the iron levels of growth-arrested worms (indicated by both calcein AM fluorescence and pftn-2:: GFP), but did not rescue growth, indicating that En in (A)the effect of t on the host iron pool is unlikely to be due to the indirect effect of the slower growth rate of worms. (E) calcein-AM fluorescence intensity of worms fed live entera or entF alone increased, indicating that the benefit of Ent on increased host iron levels is not limited to the feeding conditions graphically represented in (a). (F) Showing the introduction of FeCl₃The addition of wild-type e.coli sources, which is expected to inhibit Ent production, inhibits the growth of worms fed heat-inactivated e.coli. However, this growth is largely restored by the Ent supplementation. A representative worm image is shown in fig. D. (G) Under iron deficiency conditions treated with CaEDTA, the worms showed growth retardation. calcein-AM fluorescence of the worms decreased (increased iron levels) with FeCF supplementation (in a dose-dependent manner) or Ent supplementation. The effect of end supplementation on the decrease in fluorescence levels is equivalent to supplementing the food with 10ul of FeCl₃(175ug/ul) and "n" is the number of worms assessed. Data are presented as mean ± SEM. P<0.00 l. All data represent at least three independent experiments.

FIG. 3: bacterial enterobacterin binds to the alpha subunit of ATP synthase. (A) Schematic of the procedure for identifying an Ent binding protein from whole worm lysates by affinity chromatography using biotin-conjugated Ent. The retained proteins were identified by mass spectrometry. Two proteins identified in two independent experiments are indicated. (B) Feeding condition sketches, microscope images and bar charts showing the unsuccessful rescue of growth of animals treated with atp-1(RNAi) by end supplementation are shown. ctl-2RNAi did not alter the benefits of end supplementation. (C) For in vivo testing of Ent binding to ATP-1. The interacting proteins were pulled down from whole worm lysates using biotin-Ent, followed by streptavidin bead purification. Western blot analysis using anti-ATP 5Al antibody (antibody specificity see FIG. 10A) to detect ATP-1 in IP. (D-E) in vitro test for the binding of Ent to ATP-1. His-tagged protein binds to biotin-Ent (D), and the binding is increased by increased protein concentration and decreased by the addition of excess non-biotin-tagged Ent (E). (F) Shows Ent-mediated Fe in iron binding assays³⁺Sketches and bars of the interaction with ATP-1. With 55FeCl₃+/-Ferrophore (Ent, ferrichrome or fluorescein) treatment of whole worm lysates followed by immunoprecipitation with anti-ATP 5A 1. The relative iron levels were determined by measuring radioactivity. The presence of Ent increased 55Fe associated with ATP-1-IP by more than 10-fold. Data are presented as mean ± SEM. P<0.00 l. All data represent at least three independent experiments, except D and E (two independent experiments).

FIG. 4: ent requires ATP-1, rather than ATP synthase, in the promotion of host iron squaring. (A-D) feeding condition sketch, calcein-AM staining fluorescence image and quantitative data bar chart depicting the effect of feeding conditions on host iron levels. (A) Host iron levels decreased in ATP-1 loss-of-function (lf) homozygous animals (100% L1 arrest, n >50) under conventional feeding conditions, and the decrease in iron levels, but not growth arrest (100%, n >50), was effectively inhibited by expression of ATP-binding deficient ATP-1 mutant proteins from the transgene [ Prpl28:: ATP-1(del) ]. Data are presented as mean ± SEM. (B) RNAi knockdown of ATP-f, but not each of the other three subunits of ATP synthase, results in a decrease in the iron levels of the worms under conventional feeding conditions. Data are presented as mean ± SD. (C) Pretreatment of animals with ATP-1RNAi abolished the benefit of end supplementation when animals were fed with entF mutant bacteria, suggesting that the effect of end is dependent on ATP-1. Data are presented as mean ± SEM. (D) Pretreatment of animals with atp-1RNAi abolished the benefit of end supplementation when animals were fed heat-inactivated bacteria alone. Data are presented as mean ± SEM. (E) In an in vitro binding assay, deletion of 8aa (drqtgkta) of the ATP-binding domain did not alter ATP-1 binding to Ent, similar to that in fig. 3D. "n" is the rated number of worms. P <0.00 l. All data represent at least three independent experiments.

FIG. 5: Ent-ATP-1 interactions in mitochondria promote increased iron levels in mitochondria. (A) From a sketch of the in vivo mitochondrial iron uptake assay and data. Feeding 55FeCl to worms₃+/-Ent. Mitochondria were extracted from these worms and the relative 55Fe levels between the two samples were determined for each RNAi treatment. The presence of Ent causes an approximately 3-fold increase in mitochondrial 55Fe levels, and the Ent effect is due to the RNAi of atp-fRNAi ablation of non-other ATP synthase genes. (B) CAS staining assay showing significantly lower mitochondrial siderophore levels from worms fed entF mutant bacteria. (C) atp-1RNAi results in a decrease in mitochondrial siderophore levels. (D) In vitro mitochondrial iron uptake assay. Mitochondria were first purified from worm lysates and then treated with 55FeCl₃+/-Ent incubations and relative 55Fe levels between the two samples were measured for each RNAi treatment. The presence of Ent resulted in a 10-fold higher 55Fe level in mitochondria, and this effect was significantly reduced by RNAi of ap-1, but not by other ATP synthase genes. (E and F) Ent supplementation results in an increased activity of the enzyme containing the Fe-S cluster, as indicated by the increased activity of mitochondrial aconitase (E) and succinate dehydrogenase (F) from worms fed with food lacking Ent. P<0.05、**P<0.0l、***p<0.00 l. Data are presented as mean ± SD. All data represent at least three independent experiments.

FIG. 6: ent also promotes mitochondrial iron levels in mammalian cells by interacting with the alpha subunit of ATP synthase. (A) It was shown that end supplementation resulted in CAS staining with increased siderophore levels in HEK293T cells. Data are presented as mean ± SD. (B) In vivo Ent-biotin pull-down assays using total protein extracts from cultured human HEK293T cells +/-biotin-Ent and western blots identified ATP5a1 as an Ent binding protein. (C) In vitro test for the binding of Ent to mammalian ATP5a 1. His-tagged proteins bind biotin-Ent and the binding is outweighed by the excess non-biotin-tagged Ent. (D) Bar graph showing Ent mediated interaction between ATP5a1 and iron. HEK293T Whole cell lysate with 55FeCl₃+/-Ent treatment, followed by immunoprecipitation with anti-ATP 5A1 and measurement of radioactivity. Data are presented as mean ± SD. (E) Results of the in vivo mitochondrial iron uptake assay (similar to that for caenorhabditis elegans in fig. 5A) show that end supplementation significantly increased Fe³⁺Uptake into mitochondria and the increase is abrogated by siRNA knockdown of ATP5a 1. The effectiveness of the siRNA is shown in fig. 13A. Data are presented as mean ± SD. (F) Showing ATP5A 1-dependent effects of Ent on mitochondrial iron uptake by HEK293T cellsResults of in vitro mitochondrial iron uptake assay. As in caenorhabditis elegans (fig. 5D), the addition of Ent increased iron uptake into mitochondria, and this benefit was dramatically reduced by siRNA knockdown of ATP5a 1. Data are presented as mean ± SD. (G) Fluorescence images and quantitative data of HEK293T cells stained with the fluorescent mitochondrial iron indicator RPA. End supplementation resulted in a significant reduction in staining (thereby indicating an increase in iron), which was eliminated by knock-out of ATP5a 1. Data are presented as mean ± SEM. P<0.0l、***P<0.00 l. All data represent at least three independent experiments.

FIG. 7: a new paradigm is proposed for the iron "tug of war" between commensal bacteria and host animals. (A) The discovery of the role of lipoprotein 2(lcn2) has led to the classic concept of iron "tug-of-war" between pathogenic bacteria and the host immune system. After infection, lcn2 was induced with Ent-Fe³⁺Binding, this blocks the effect of Ent in taking iron from the host cell for bacterial growth (Baumler and Sperandio, 2016; elermann and Arthur, 2017; Xiao et al, 2017). This chelating function inhibits bacterial growth, but may not be conducive to host iron homeostasis and other physiological effects. (B) The surprising beneficial effect of the Ent-ATP synthase alpha subunit in promoting mitochondrial iron concentration points to a new mechanism that evolved to offset the known negative effects of Ent on iron homeostasis and thereby enhance the symbiotic relationship between intestinal bacteria and animals.

FIG. 8: the bacterium enterobactin promotes caenorhabditis elegans development. (A) It is shown that worms that bind to either the entera or the entF mutant e.coli fed heat inactivated e.coli grew slower and this defect was fully suppressed by the Ent supplementation in feeding condition sketches and microscopic images. Quantitative data is shown in fig. 1D. B) On the outer membrane of bacteria to facilitate uptake of Ent-Fe in E.coli³⁺A sketch of the complex's iron enterobactin receptor FepA. (C) Feeding condition draft and microscopy images showing that feeding of heat inactivated e.coli worms in combination with fepA mutant e.coli, unlike feeding with either the entera or the entF mutant, did not show growth defects. Quantitative data are shown in fig. 1F. (D) The entA and entF mutant E.coli strains showed growth rates similar to those of the parent wild type strain E.coli K12-BW 25113.(E) The E.coli strains of the entA and entF mutants colonize the host gut as efficiently as the parental wild-type strains. (F) Feeding condition draft, microscopic image and bar graph showing that neither fluorescein nor ferrichrome resulted in significant growth defects in worms fed heat-inactivated food plus wild-type live e. (G) Fluorescence microscopy of worms containing mtGFP under the same feeding conditions as in (F). Supplementation with each of the three siderophores did not affect mitochondrial morphology in the assay system of the invention. (H) In liquid culture, the siderophore fluorescent siderophore produced by pseudomonas aeruginosa (p. aeruginosa) is toxic to worms because it damages host mitochondria (mtGFP network pattern fragmentation and reduction to large blobs) (Kirienko et al, 2015). However, Ent does not disrupt mitochondrial morphology. (I) The feeding condition sketch, microscopic image and bar graph show that supplementation of heat-inactivated e.coli OP50 with Ent failed to rescue host development, demonstrating the view that: supporting worm growth requires metabolites produced by a variety of bacteria from live bacteria (Qi et al, 2017). "n" is the rated number of worms. Data are presented as mean ± SEM. P<0.000l。

FIG. 9: functional relationship between Ent, iron concentration and worm growth. (A) Shows that more Fe is added to heat-inactivated food³⁺(FeCl₃) Feeding condition sketch, microscope image and bar graph without inhibition of growth defect (a) caused by end deficiency (see fig. 1B). Data are presented as mean ± SD. (B) Shows that more Fe is added unlike the Ent supplement³+ no calcein-AM staining of the iron levels increased in worms fed with heat-inactivated e.

Data are presented as mean ± SEM. (C) Adding more heme to food did not inhibit growth defects caused by Ent deficiency. Data are presented as mean ± SEM. (D) Microscope images showing that adding more ferric trichloride to wild-type e.coli in the new assay system inhibited worm growth. Growth defects were recovered by end supplementation. The quantitative data is shown in figure 2F. "n" is the rated number of worms.

FIG. 10: in vitro mapping of the Ent binding sequence of ATP-1. (A) A single band was detected in whole worm extracts with an antibody against the mammalian ATP synthase alpha subunit, and the band intensity was greatly reduced in ATP-1(RNAi) -treated samples, demonstrating the specificity of this antibody for the worm protein ATP-1. (B-C) in vitro test for binding between iron-bound Ent and ATP-1. Increasing the concentration of His-tagged protein increased binding to biotin-Fe-Ent (B). The binding is reduced by adding an excess of non-biotin-labeled Ent (C). Data are presented as mean ± SEM. (D) The full-length sequence of the ATP-1 protein was divided into three segments and then expressed in E.coli. In vitro binding assays using purified proteins showed that the middle segment retained Ent binding capacity. (E) Eight peptides covering the middle segment of ATP-1 (identified in D) were tested for binding, revealing that the 21 amino acid peptide (FCIYVAVGQKRSTVAQIVKRL) was sufficient to bind Ent in an in vitro binding assay. (F) The ATP-1 protein lacking the 21 amino acid sequence loses the Ent binding ability. Thus, even though this 21-residue peptide is deficient for its iron uptake function, it is both essential and sufficient for end binding.

FIG. 11: binding of ATP-1 to Ent is independent of the β subunit of ATP synthase and sequence comparison of ATP-1 to human ATP5A 1. (A) Immunostaining showing co-localization of the alpha subunit with the ATP synthase beta subunit in HEK293T cells. (B) atp-1(RNAi) showed a slow growth phenotype in C.elegans. (C) Western blot showing that binding of ATP-1 to Ent is independent of the β subunit of ATP synthase (ATP-2). Biotin-Ent was fed to worms treated with control or atp-2RNAi and total protein extracts were isolated and then subjected to streptavidin bead purification. ATP-1 protein was detected in both samples by Western blotting using antibodies against the alpha subunit of ATP synthase. The worms grew more slowly after atp-2(RNAi) treatment, indicating that RNAi knockdown atp-2 was effective. (D) Alignment of protein sequences from the alpha subunits of C.elegans and human ATP synthase. The predicted ATP and Ent binding sites are indicated.

FIG. 12: ATP-1 co-localizes with MitoTracker. An image of a dissected intestinal immunostain showing co-localization of ATP-1 with MitoTracker. The atp-1RNAi treatment resulted in a reduction in immunostaining.

FIG. 13: the siRNA effectively reduced the level of ATP5a 1. ATP5a1 protein levels were reduced in cells treated with siRNA ATP5a 1.

FIG. 14: mice grew slowly with colonization by the entF bacteria. 5-week-old sterile mice were colonized with wild-type or entF (enterobactin-deficient) bacteria. After colonization, mice were grown for 4 weeks. Body weight was measured weekly and weight gain was calculated.

FIG. 15: the 2-D chemical structure of enterobactin. (the coordinated oxygen atoms are indicated in red).

FIG. 16: enterobacterin and synthetic analogs thereof: catecholases TRENCAM and salicylate SERSAM, SER (3M) SAM, tressam and TREN (3M) SAM ligands (coordinated oxygen atoms are indicated in red).

FIG. 17: a schematic diagram showing exemplary escherichia coli microorganisms in the intestinal lumen secreting a ferric binding compound Ent taken from g.j.anderson. "iron competition-host counterattack" new england medical journal, 2018, 11 months and 22 days.

FIG. 18: in media with iron chelators, end addition increased iron uptake in human HEK293 cells. When the iron chelator Desferrioxamine (DFO) was added to the medium, the iron levels in the cells were significantly reduced as indicated by an increase in calcein AM staining fluorescence. By adding Ent (1.5 μ M) to the medium, the iron level was substantially restored. The decrease in calcein AM fluorescence with the addition of Ent (45%) was stronger than the test without DFO.

FIG. 19: ent and ATPS α promote iron transport across the lipid bilayer of the liposome. (A) A sketch of experimental conditions and a graphical quantification of calcein AM staining. Calcein AM dye was added to the liposomes +/-ATPS α and the fluorescence intensity of the liposomes was measured. (B) A sketch of experimental conditions and a graphical quantification of iron uptake. Combining liposomes with radiolabeled Fe³⁺(⁵⁵FeCl₃) Incubate and measure the radioactivity (relative CPM) of the liposomes.

FIG. 20: end supplementation by oral gavage promoted increased hemoglobin and spleen iron levels in an anemic mouse model (dietary anemia). Iron Deficiency Diet (IDD) (or control diet) was fed to 3-week-old female mice for 6 weeks to induce anemia (Tong-Tong)As confirmed by a per-hemoglobin measurement). Then +/-Ent (two concentrations) or +/-FeSO by oral gavage (once every two days) over a two week period₄Mice (5/group) were treated.

FIG. 21: ent supplemented by drinking water (ad libitum) promoted an increase in hemoglobin levels in an anemic mouse model (dietary anemia). Mice at 3 weeks of age were fed an Iron Deficiency Diet (IDD) (or control diet) to induce anemia. The mice were then fed IDD +/-Ent added to drinking water for another two weeks. Fresh dilutions of Ent in water were provided once a week.

FIG. 22: ent supplemented by drinking water (ad libitum) promoted the growth of mice fed a control (iron-replete) diet. Male mice of 4.5 weeks of age were treated with the control iron-rich Control Diet (CD) matched to the iron-deficient diet (IDD) used in fig. 20 and 21.

FIG. 23: ent promotes the growth of mice colonized with a single strain of e. In addition to fig. 14, the inventors of the present invention exhibited (a). Five-week-old female sterile (GF) mice were colonized with a single non-pathogenic e.coli (K12) strain, wild-type or entF. Mouse growth (weight gain) was measured over the next 4 weeks. Sterile (GF) mice colonized with entF e. Interestingly, the difference in weight gain was greatest during the first two weeks after colonization. (B and C) iron levels in late mouse were only significantly lower in spleen (about 35%) but not in liver or other tissues, consistent with the results seen in fig. 20. The iron level was measured. (D) The Ent supplementation overcomes the growth delay of GF mice colonized with entF e. GF female mice colonized with entF bacteria were supplemented with Ent [2 concentrations were added to drinking water (pH 5.5) once weekly ].

FIG. 24: the effect of enterobactin (Ent) in promoting animal development was not seen in other siderophores. Newly hatched larvae of caenorhabditis elegans are fed with wild-type K12 e.coli or entF e.coli supplemented with the indicated siderophore.

FIG. 25: ent stability test under different solvent and pH conditions. (A) By CAS liquid assay (adapted from Arora)&Verma 2017) measureEnt stability. Degradation is indicated by increased absorbance. At H₂Ent diluted in O (pH 5.5) (with 10% DMSO) degraded rapidly within the first 60 minutes. In contrast, Ent diluted in 100% DMSO showed little change during the assay period. (B) H at different pH was measured by CAS liquid assay₂Ent stability in O. At H₂Diluted Ent in O (pH 6.5/7) to acidic and basic H₂The other dilutions in O are more stable. (C) Ent stability relative to Fe-Ent was measured by NGAL fluorometry (adapted from Goetz et al, 2002) and graphically represented by a linear trend line. Ent and Fe-Ent were prepared in the same buffer at the same concentration. Degradation is indicated by increased relative fluorescence. Fe-Ent is more stable than Ent alone. The diluent was kept at room temperature and exposed to light for all tests. The error is the standard deviation of the mean.

Detailed Description

The present invention may comprise novel systems, methods and compositions for the therapeutic administration of Ent and/or an Ent analog to treat iron deficiency in a subject. As indicated above, fig. 15 is a typical siderophore enterobactin (Ent) biosynthesized from gram-negative species of Enterobacteriaceae (Enterobacteriaceae) containing Escherichia coli (e.coli), salmonella and klebsiella. Decades of exploration regarding the biosynthesis and coordination chemistry of enterobacterin, as well as protein studies involved in the cellular transport and processing of enterobacterin, have provided a detailed molecular and physiological understanding of how this chelate contributes to bacterial iron homeostasis and colonization (Raymond et al, Proc. Natl.Acad. Sci.U.S.A.) (2003, 100, 3584) -3588). The enterobacterin synthase contains four proteins, EntB, EntD, EntE, EntF and is responsible for the production of enterobacterin from L-serine and 2, 3-dihydroxybenzoic acid (DHB). After biosynthesis, Ent is exported to it for Fe scavenging³In the extracellular space of (3). Enterobacterin is reacted with Fe via its three catecholase groups under a Ka of about 1049M-1³And (4) coordination.

For example, one aspect of the present invention relates to a method of treating iron deficiency and preferably iron deficiency anemia in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of Ent and a pharmaceutically acceptable carrier thereof and/or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition thereof. In this embodiment, the Ent may comprise purified, substantially purified, and/or isolated Ent, which may be further combined with a pharmaceutically acceptable composition, such as an excipient.

Another aspect of the invention relates to a method of preventing iron deficiency and preferably iron deficiency anemia in a subject in need thereof, said method comprising administering to the subject a prophylactically effective amount of Ent and/or an Ent analogue and a pharmaceutically acceptable carrier thereof and/or a pharmaceutically acceptable salt thereof and/or a pharmaceutical composition thereof.

In another example, one aspect of the invention relates to a method of treating iron deficiency in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Ent by introducing a probiotic and/or a symbiotic delivery vehicle. In another example, one aspect of the invention relates to a method of treating iron deficiency in a subject in need thereof, the method comprising administering to the subject a prophylactically effective amount of Ent by introducing a probiotic and/or a symbiotic delivery vehicle.

For example, one aspect of the present invention relates to a method of treating iron deficiency and preferably iron deficiency anemia in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of Ent or an analogue thereof by a non-pathogenic symbiotic and/or probiotic. In this embodiment, the invention may comprise genetically modified non-pathogenic commensal donor bacteria and/or probiotic donor bacteria that may be configured to express and/or overexpress Ent en in a recipient host. In one embodiment, the genetically modified non-pathogenic commensal donor bacterium and/or probiotic donor bacterium can be configured to express and/or overexpress one or more genes involved in Ent biosynthesis. For example, in this preferred embodiment, one or more genes involved in the biosynthesis of Ent may be part of an expression cassette and further operably linked to one or more expression control sequences. In a preferred embodiment, this promoter may be a constitutive promoter.

In one embodiment, one or more of the genetically engineered probiotics and/or symbionts mentioned above may be part of a pharmaceutical and/or nutraceutical composition. In further embodiments, the isolated Ent and/or one or more of the genetically engineered probiotics and/or symbionts mentioned above may be part of a food or beverage additive that may be administered a therapeutically effective amount to treat a disease condition. In another embodiment, the isolated Ent and/or one or more of the genetically engineered probiotics and/or symbionts mentioned above may be part of a supplement and may be further coupled with additional dietary supplements such as a dietary iron supplement.

A nucleotide sequence or said polynucleotide sequence is "operably linked to" one or more expression control sequences (or e.g., a promoter and optionally an enhancer) when the expression control sequences control and regulate transcription and/or translation of the polynucleotide sequence. As used herein, the phrase "gene product" refers to an RNA molecule or protein.

Furthermore, the term "gene" may sometimes refer to a gene sequence, a transcribed and possibly modified mRNA of said gene or a translated protein of said mRNA.

Examples of such Ent biosynthetic genes may include entB, entD, entE and/or entF. Additional embodiments may include genes involved in the biosynthesis of the Ent precursor, including entC, entB, and entA. Such genes may be heterologous and or endogenous to the subject and comprise all homologues and orthologues of the gene. It should be noted that the nucleic acid and amino acid sequences of the above-mentioned genes are within the knowledge of one of ordinary skill in the art and are expressly incorporated herein by reference. As used herein, the term "probiotic" generally refers to bacteria that can colonize a target host for a time sufficient to deliver a therapeutically effective amount of Ent to the host.

The terms "purified," "substantially purified," and "isolated" refer to compounds useful in the invention that are free of other dissimilar compounds with which they are typically associated in their natural state such that the compounds make up at least 0.5, 1, 5, 10, 20, 50, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 99.9 weight percent of the mass of a given sample or composition. In one embodiment, these terms refer to a compound that constitutes at least 95%, 98%, 99%, or 99.9% by weight of the mass of a given sample or composition.

The term "pharmaceutically acceptable salt" refers to salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al describe in detail in journal of pharmaceutical Sciences (J. pharmaceutical Sciences), 1977,66,1-19, which is incorporated herein by reference.

Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Salts may be prepared during the final isolation and purification of the compound or separately by reacting the appropriate compound in its free base form with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, acetate,

maleate, malonate, DL-mandelate, mesitylene sulfonate, methanesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalic acidSalts, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphonate, picrate, pivalate, propionate, pyroglutamate, succinate, sulfonate, tartrate, L-tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate (p-tosylate) and undecanoate. In addition, the bases in the compounds disclosed herein may be quaternized with: methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate; decyl, lauryl, myristyl and sterol chlorides, bromides and iodides; and benzyl and phenethyl bromides. Examples of acids that may be used to form therapeutically acceptable salts include: inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid; and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. "base addition salts" refers to salts derived from the appropriate bases, including alkali metal, alkaline earth metal, and quaternary ammonium salts. Thus, the present invention contemplates sodium, potassium, magnesium, and calcium salts, and the like, of the compounds disclosed herein. Base addition salts can be prepared during the final isolation and purification of the compounds, usually by reacting the carboxyl group with a suitable base (such as the hydroxide, carbonate or bicarbonate of a metal cation) or with ammonia or an organic primary, secondary or tertiary amine. The cation of the therapeutically acceptable salt comprises: lithium, sodium (by using, for example, NaOH), potassium (by using, for example, KOH), calcium (by using, for example, Ca (OH))₂) Magnesium (by using, for example, Mg (OH)₂And magnesium acetate), zinc (by using, for example, Zn (OH)₂And zinc acetate) and aluminum; and nontoxic quaternary ammonium cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine (procaine), dibenzylamine, N-dibenzylphenethylamine, l-phenamine (l-ephenamine), and N, N-dibenzylethylenediamine. Other representative organic amines useful for forming base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, hydroxidCholine, hydroxyethyl morpholine, hydroxyethyl pyrrolidone, imidazole, N-methyl-d-glucamine, N ' -dibenzylethylenediamine, N ' -diethylethanolamine, N ' -dimethylethanolamine, triethanolamine and tromethamine. Basic amino acids (e.g., 1-glycine and 1-arginine) and amino acids that are zwitterionic at neutral pH (e.g., betaine (N, N-trimethylglycine)) are also contemplated.

The term "administering" refers to injecting, implanting, absorbing, ingesting Ent that may be part of a pharmaceutical composition or ingesting a probiotic configured to produce Ent as described herein or a probiotic in a pharmaceutical composition thereof.

The terms "treat", "treating" and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a "pathological condition" described herein (e.g., a disease, disorder or condition, or one or more signs or symptoms thereof). In some embodiments, treatment may be administered after one or more signs or symptoms are present or observed. In other embodiments, the treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., based on symptom history and/or based on genetic or other susceptibility factors). Treatment may also be continued after a minor withdrawal of symptoms, for example to delay or prevent relapse. In a preferred embodiment, the treatment may be for iron deficiency related disorders, such as iron deficiency anemia.

A "therapeutically effective amount" of a compound of the invention, preferably Ent or an Ent analog, or a pharmaceutical composition thereof, is an amount sufficient to provide a therapeutic benefit in treating a disease or delaying or minimizing one or more symptoms associated with a condition. A therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in treating a condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a condition, and/or enhances the therapeutic efficacy of another therapeutic agent. A "therapeutically effective amount" may also mean an amount of a compound of the invention that is "prophylactically effective" sufficient to prevent the disease or one or more symptoms associated with the condition, or to prevent recurrence thereof. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a prophylactic benefit in preventing a condition. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

The pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology. Generally, such a preparation method comprises the following steps: the compound Ent or Ent analog or Ent conjugate or probiotic configured to produce and or overproduce Ent of Ent (i.e., "active ingredient") is associated with a carrier or excipient and/or one or more other auxiliary ingredients and then the product is shaped and/or packaged, if needed and/or desired, into the desired single or multiple dosage units. The pharmaceutical or nutraceutical composition may be manufactured, packaged and/or sold in bulk as a single unit dose and/or as a plurality of single unit doses. A "unit dose" is a discrete amount of a pharmaceutical composition that includes a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to the subject and/or a convenient fraction of such dose, for example half or one third of such dose.

The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and/or any additional ingredients in the pharmaceutical compositions of the invention will vary depending on the identity, size, and/or condition of the subject being treated and further depending on the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surfactants and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricants, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dicalcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium carboxymethyl starch, clays, alginic acid, guar gum, citrus pulp, agar-agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly (vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium carboxymethyl starch), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (vegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

Exemplary surfactants and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, carrageenan (chondlux), cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and magnesium aluminosilicate), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin, monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan (carrageenans), cellulose derivatives (e.g., sodium carboxymethylcellulose, sodium alginate, xanthan gum, pectin, and magnesium aluminosilicate), long chain amino acid derivatives, high molecular weight alcohols (e.g., sodium carboxymethylcellulose, sodium alginate, and magnesium silicate, sodium alginate, and magnesium silicate, sodium alginate, and magnesium silicate, sodium alginate, and magnesium silicate, sodium alginate, magnesium silicate, and magnesium silicate, Powdered cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate: (A)

20) Polyoxyethylene sorbitan

60) Polyoxyethylene sorbitan monooleate (A)

80) Sorbitan monopalmitate (A)

40) Sorbitan monostearate (C)

60) Sorbitan tristearate (C)

65) Glycerol monooleate, sorbitan monooleate (A)

80) Polyoxyethylene esters (e.g., polyoxyethylene monostearate) ((C))

45) Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxyl stearate and

) Sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.,

) Polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether: (A)

30) Poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, and sodium lauryl sulfate,

F-68, pomadePoloxamer (Poloxamer) P-188, cetyltrimethylammonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.

Exemplary binding agents include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, and the like), natural and synthetic gums (e.g., acacia gum, sodium alginate, carrageenan extract, panrag gum (panwar gum), ghatti gum (ghatti gum), cyperus shell mucilage (mucina of isapol husks), carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly (vinyl-pyrrolidone), magnesium aluminum silicate ((r) (starch and starch paste), and the like

And larch arabinogalactans), alginates, polyethylene oxides, polyethylene glycols, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohols and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcoholic preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium sodium edetate, dipotassium edetate, etc.), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine (hexetidine), imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin a, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopheryl acetate, dexemethylamine mesylate, cetyltrimethylammonium bromide, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), ethylenediamine, Sodium Lauryl Sulfate (SLS), Sodium Lauryl Ether Sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, sodium hydrogen sulfite, sodium hydrogen sulfite, sodium hydrogen, sodium hydrogen sulfite, sodium hydrogen, sodium hydrogen sulfite, sodium hydrogen, sodium hydrogen, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen sulfate, sodium hydrogen,

Plus、

Methyl p-hydroxybenzoate,

115、

II、

And

exemplary buffers include citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium valerate (levulinate), valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate (calcium hydroxide phosphate), potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and mixtures thereof.

Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant seed oil, borage oil, juniper oil, chamomile oil, canola oil, caraway oil, palm wax oil, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, cucurbit oil, grapeseed oil, hazelnut oil, hyssop oil, isopropyl myristate oil, jojoba oil, macadamia nut oil, lavandin oil, lavender oil, lemon oil, litsea cubeba oil, waiian oil, mallow oil, mango butter, mink oil, nutmeg oil, olive oil, orange rouge oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, melon seed oil, rapeseed oil, canola oil, palm kernel oil, sesame seed oil, and peanut oil, Rice bran oil, rosemary oil, safflower oil, sandalwood oil, camellia oil, savory oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, cedrela sinensis oil, vetiver oil, walnut oil and wheat germ oil. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs, preferably containing a unit dose of Ent or a unit dose of a probiotic configured to express or overexpress Ent. In addition to the active ingredient, the liquid dosage forms may include inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates of the invention are combined with a solubilizing agent, e.g., a solubilizer

Alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that can be employed are water, ringer's solution, u.s.p. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material that is poorly water soluble. The rate of absorption of the drug then depends on its rate of dissolution, which in turn may depend on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form may be achieved by dissolving or suspending the drug in an oily vehicle.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active ingredient is mixed with: at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binding agents, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents, such as paraffin; (f) absorption promoters, such as quaternary ammonium compounds; (g) humectants, such as cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may contain buffering agents.

Solid compositions of a similar type may be employed as fillers in soft-filled gelatin capsules and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmacological arts. The solid dosage form may optionally include an opacifying agent and may be a composition that: the solid dosage form may release one or more active ingredients only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of encapsulation compositions that may be used include polymeric materials and waxes. Solid compositions of a similar type may be employed as fillers in soft-filled gelatin capsules and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The active ingredient may be in microencapsulated form together with one or more excipients as indicated above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active ingredient may be mixed with at least one inert diluent, such as sucrose, lactose or starch. In accordance with common practice, such dosage forms may include, in addition to the inert diluent, additional substances such as tableting lubricants and other tableting aids, for example magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may include buffering agents. The solid dosage form may optionally include an opacifying agent and may be a composition that: the solid dosage form may release one or more active ingredients only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of encapsulating agents that may be used include polymeric substances and waxes.

The compounds and compositions provided herein can be administered by any route, including enterally (e.g., orally), parenterally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, subcutaneously, intraventricularly, transdermally, intradermally, rectally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, cream, and/or drops), mucosally, nasally, buccally, or sublingually; administration by intratracheal instillation, bronchial instillation and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes of administration for the compounds and compositions disclosed herein are inhalation and intranasal administration, subcutaneous administration, mucosal administration, and intradermal administration. The most appropriate route of administration will generally depend on various factors, including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract) and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).

The exact amount of "active ingredient" required to achieve an effective amount will vary from subject to subject, depending on, for example, the species, age, and general condition of the subject, the severity of the side effect or disorder, the nature (identity) of the particular compound, the mode of administration, and the like. The desired dose may be delivered three times a day, twice a day, once a day, every other day, every third day, every week, every second week, every third week, or every fourth week. In certain embodiments, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations) can be used to deliver a desired dose.

In certain embodiments, an effective amount of a compound for administration one or more times a day to a 70kg adult human may include from about 0.0001mg to about 3000mg, from about 0.0001mg to about 2000mg, from about 0.0001mg to about 1000mg, from about 0.001mg to about 1000mg, from about 0.01mg to about 1000mg, from about 0.1mg to about 1000mg, from about 1mg to about 100mg, from about 10mg to about 1000mg, or from about 100mg to about 1000mg of the compound per unit dose.

Kits (e.g., pharmaceutical packs) are also encompassed by the invention. The provided kits can include the compound Ent or a composition (e.g., a pharmaceutical composition or a diagnostic composition) and a container (e.g., a vial, ampoule, bottle, syringe and/or dispenser package or other suitable container). The provided kits can include an antibody that selectively binds to an enterobacterin or a composition (e.g., a pharmaceutical composition or a diagnostic composition) and a container (e.g., a vial, ampoule, bottle, syringe, and/or dispenser package or other suitable container). In some embodiments, the provided kits may optionally further comprise a second container comprising an excipient (e.g., a pharmaceutically acceptable excipient) for diluting or suspending a pharmaceutical composition or compound of the invention. In some embodiments, the compound Ent or the composition provided in the first and second containers are combined to form one unit dosage form. In another aspect, the invention provides a kit comprising a first container comprising an antibody produced using compound Ent, i.e., an antibody that selectively binds enterobacterin.

The term "subject" refers to any animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human (e.g., a man, a woman, or a child). A human may be of any gender and may be at any stage of development. In certain embodiments, the subject has been diagnosed with a condition or disease to be treated. In other embodiments, the subject is at risk for developing a condition or disease. In certain embodiments, the subject is an experimental animal (e.g., a mouse, rat, rabbit, dog, pig, or primate). The experimental animal may be genetically engineered. In certain embodiments, the subject is a domestic animal (e.g., a dog, cat, bird, horse, cow, goat, sheep, or chicken).

Examples of the invention

Example 1: bacterial mutant screening identifies the benefits of enterobacterin produced by e.coli on host development.

In one embodiment of the present technology, the inventors of the present invention created a unique assay for facilitating the identification of microbial metabolites beneficial for the growth and development of a host animal. Previous studies by the present inventors revealed that heat-inactivated (HK) escherichia coli lacks certain molecules that are commonly required for caenorhabditis elegans larvae to grow. Larval growth recovered when the HK e.coli plates were supplemented with trace amounts of live e.coli, which the HK e.coli alone could not support worm growth (fig. 1A), indicating that trace amounts of live bacteria produced metabolites that made the HK food available. This unique feeding condition was used to find E.coli mutants that could potentially fail to support normal helminth growth because they could not provide specific metabolites to benefit the host animal. After screening of the E.coli single gene knock-out library (Keio collection) of E.coli, the inventors of the present invention found that the worm growth of any E.coli mutant in trace amounts among five E.coli mutants whose enterobactin (Ent) biosynthesis was disrupted was significantly slower (FIG. 1B, C). Strikingly, the inventors of the present invention observed that the defect of helminth growth was completely overcome by dietary supplementation of Ent (fig. 1D and fig. 8A). In addition, supplementation with the metabolic intermediate 2,3-DHBA rescued worm growth on entero e.coli, but not on entero e.coli (fig. 1C, E), confirming that only the end product Ent can provide this benefit to the worms.

The inventors of the present invention further show that this beneficial effect of Ent on worm growth may not be related to the bacterial use of Ent as a siderophore. Specifically, disruption of fepA encoding the e.coli outer membrane receptor for iron Ent (fig. 8B) did not affect helminth development (fig. 1F and fig. 8C). The inventors of the present invention also found that the entA and entF mutant bacteria did not show significant growth defects under the outlined culture conditions or in helminth gut colonization (fig. 8D and E).

Supplementation with two other siderophores (fluorescein and ferrichrome) failed to produce a similar effect on worm growth (fig. 1G and 8F), suggesting that the observed effect of Ent is specific. Previous studies have shown that siderophores produced by P.aeruginosa are toxic to C.elegans by damaging the host mitochondria in liquid culture (Kirienko et al, 2015), raising the problem of: whether the negative consequences of fluorescent siderophiles or ferrichromes are primarily due to the toxicity of these siderophores. The inventors of the present invention thus performed tests using solid media under the identified culture conditions and observed that there was no apparent growth defect (fig. 8F) or mitochondrial morphology defect (fig. 8G) in c. In addition, the inventors of the present invention found that end supplementation did not disrupt mitochondrial morphology even in liquid culture (fig. 8H), unlike the effect of fluorescent siderophiles.

To assess siderophore levels throughout the worms, established assays (Schwyn and neillands, 1987) were modified and the inventors of the present invention found that worms fed wild type e.coli contained siderophore levels significantly higher than worms fed the entF mutant e.coli (fig. 1H). Given that high iron levels are known to inhibit Ent biosynthesis (Kwon et al, 1996), the production of Ent from e.coli under culture conditions is consistent with the relatively low iron medium in the referenced experiments.

End supplementation alone did not have any appreciable effect on the development of worms fed only with HK bacteria (fig. 81), indicating that HK bacteria are not merely deficient in end or any one of the specific metabolites (Qi et al, 2017). In contrast, worms fed large numbers of live entA or entF mutant bacteria continued to grow at a slower rate (fig. 1I), confirming that a significant benefit of Ent on host development was clearly detected by the new sensitive assay system (fig. 1 BD). This benefit of Ent on animal growth may also be evident in certain natural environments.

Example 2: bacterial enterobacterin promotes the host iron pool.

Due to Ent to Fe³⁺Has high affinity, and thus Ent can potentially benefit the growth and development of the host animal by affecting iron homeostasis. The inventors of the present invention employed the commonly used fluorescent cell-permeable dye calcein AM (whose emission is quenched by iron binding) and applied the calcein AM to live worms as described previously to estimate the overall iron level of the host. Worms fed either the entera or the entF mutant bacteria had much lower iron levels, as evidenced by greatly increased fluorescence intensity, and patch levels were restored by Ent supplementation (fig. 2A). The present inventors also examined the expression of the iron-responsive gene ftn-2 encoding the C.elegans homologue of the iron storage protein ferritin (Romney et al),2011). pfln-2. expression of GFP reporter gene was greatly reduced in helminths fed either entA or entF mutant bacteria (FIG. 2B). Thus, bacterial Ent increased the iron level of the host c.

To rule out the effect of worm growth status on the increase in iron levels promoted by Ent, the inventors of the present invention used two iron markers indicating that worms fed HK e.coli showed lower iron levels and that this defect was suppressed by Ent supplementation (fig. 2C, D), while worms remained stagnant under both conditions. In contrast, iron levels were lower in worms fed only with large numbers of live entera or entF mutant bacteria (fig. 2E), where the worms continued to grow, albeit at a slower rate (fig. 1H). Thus, the effect of Ent on host iron levels was independent of other feeding conditions and worm growth.

The results from feeding only HK food (fig. 2C, D) provide additional evidence: the beneficial Ent effect is not due to a secondary effect of bacterial use of Ent.

Based on the formulation and the fact that the biosynthesis of Ent in bacteria will be inhibited under iron-laden conditions, typical worm growth media (NGM plates) inoculated with e.coli as food appear to present a low iron environment. Adding more Fe to food³⁺(FeCl₃) Neither growth deficiency in animals fed food lacking Ent was inhibited (fig. 9A) nor did cellular iron levels increase (fig. 9B). Addition of heme also did not affect animal growth (fig. 9C). Thus, Ent promotes optimal iron uptake and caenorhabditis elegans growth, regardless of iron levels in the diet. If Ent is required for optimal caenorhabditis elegans development, the addition of more ferric chloride to wild-type E.coli in the novel assay system of the invention (FIG. 2F) is expected to inhibit Ent production in a live E.coli source and thus slow worm growth. In fact, the inventors of the present invention observed worm growth defects in the case of adding ferric trichloride to live E.coli, and this growth defect was suppressed by end supplementation (FIGS. 2F, 9D). This demonstrates that Ent is required even in iron-rich environments, which in turn may indicate that iron levels in the caenorhabditis elegans gut are typically not reached to result in the production of Ent from the gut microbiota being producedThe height of complete inhibition.

In addition, previous studies showed that under iron deficient conditions with CaEDTA treatment of the worms, the iron levels and growth rate of the worms decreased (Klang et al, 2014) (fig. 2G). However, by adding more FeCl₃Or by end supplementation, both defects are suppressed (fig. 2G). Remarkably, 10 times FeCl₃Supplementation restored the iron levels of the CaEDTA-fed worms to the levels observed in the CaEDTA plus additional Ent-fed worms (fig. 2G), indirectly suggesting that, under this condition, Ent-mediated iron uptake may be responsible for at least a 10-fold increase in iron levels by the worms. The results show that Ent has a profound effect on host iron homeostasis under iron deficiency conditions.

Example 3: bacterial enterobacterin binds to the host ATP synthase alpha subunit.

To understand the mechanism behind the effect of Ent on worm iron homeostasis, the inventors of the present invention used affinity chromatography using immobilized Ent followed by mass spectrometry to identify the Ent binding proteins of worms (fig. 3A). Only two candidate proteins CTL-2 and ATP-1 were captured in both independent experiments (FIG. 3A and Table 1). CTL-2 is a homologue of catalase known to bind iron. ATP-1 is an alpha subunit of mitochondrial ATP synthase whose role in iron biology is unknown (Junge and Nelson, 2015). The inventors then tested each protein for its need for the effect of Ent on animal growth. RNAi of the ATP-1 gene, but not CTL-2, prevented rescue of worm growth by Ent supplementation (FIG. 3B), suggesting that ATP-1, but not CTL-2, may play a key role in mediating the Ent function observed in the host.

The inventors then performed three additional tests to confirm the binding of Ent to ATP-1. First, in vivo assays, the worms were fed bacteria +/-biotin-Ent and total protein extracts were isolated, followed by streptavidin bead purification and SDS-PAGE. The ATP-1 protein was clearly detected by Western blotting using antibodies against the alpha subunit of mammalian ATP synthase (FIG. 3C and FIG. 10A), indicating the interaction between Ent and ATP-1. Second, in an in vitro binding assay, the inventors found that biotin-Ent (without iron) binds ATP-1-His efficiently (fig. 3D) and that the binding could be outweighed by excess Ent (fig. 3E). Just like the iron-free biotin-Ent, the iron-bound biotin-Ent also bound ATP-1 protein (fig. 10B, C).

Finally, the inventors of the present invention tested the ability of Ent to mediate the interaction between ATP-1 and iron. The inventors of the present invention added radiolabeled iron (55 FeCl) to helminth lysates₃) And then immunoprecipitating ATP-1. By measuring radioactivity in ATP-1-IP samples, the inventors of the present invention found that the addition of Ent (but not the other two siderophores) greatly increased ATP-1 binding to 55Fe (fig. 3F), demonstrating the specific role of Ent in mediating the interaction between ATP-1 and iron. Additional analysis showed that the 21 amino acid sequence of ATP-1 plays a key role in the binding of Ent (FIGS. 10D-F). (notably, with respect to FIG. 11D, the C.elegans ATP-1 amino acid sequence is identified herein as SEQ ID No.7, while the human ATP-1 amino acid sequence is identified herein as SEQ ID No. 8.) together these results indicate that bacterial Ent binds directly to eukaryotic ATP-1, which promotes the interaction of ATP-1 with iron.

Example 4: ATP-1 is required to promote host iron levels in an enterobacterin-dependent manner.

The inventors next tested whether Ent promoted the host iron pool by the Ent-ATP-1 complex. The inventors of the present invention first determined the role of ATP-1 in iron homeostasis by demonstrating a large reduction in iron levels in ATP-1 loss of function (lf) c.elegans mutants or wild-type worms treated with ATP-1(RNAi), as indicated by an increase in calcein-AM fluorescence (fig. 4A, B). The inventors then determined Ent

The effect in promoting the iron horizontal surface of the worms was dependent on ATP-1, since RNAi of ATP-1 abolished the iron level gain seen in the case of end supplementation of the entF mutant bacteria (fig. 4C). It was also found that the effect of HK E.coli +/-Ent on growth-arrested animals (FIG. 2C) was also dependent on atp-1 (FIG. 4D). Finally, host iron levels were not significantly altered by RNAi knockdown of each of the other three subunits of ATP synthase (fig. 4B), suggesting that the effect of ATP-1 on iron levels is unlikely due to an indirect effect that disrupts ATP synthase function. Thus, bacterial Ent promotes host iron homeostasis through its interaction with the ATP synthase alpha subunit.

Example 5: the observed ATP-1 function is independent of its role in ATP synthase.

Like the ATP synthase alpha subunit, ATP-1 is expected to interact with the beta subunit and other subunits of this large enzyme complex. Using immunostaining, the inventors of the present invention observed co-localization of the alpha (ATP5a1) and beta (ATP5B) subunits of mammalian ATP synthase in mitochondria (fig. 11A). Consistent with the requisite role of ATP synthase, loss-of-function mutations in both ATP-1 and alp-2 showed the L1 arrest phenotype of C.elegans (FIG. 4A). RNAi of either atp-1 or atp-2 also showed growth defects (FIG. 11B, C). Thus, it was tested whether ATP-1 functions in promoting the iron pool of the host depending on the other subunits of ATP synthase. It was found that binding of ATP-1 to Ent was not affected by RNAi of the β subunit of ATP synthase (ATP-2) (fig. 11C), and as indicated in fig. 4B, no decrease in iron levels caused by ATP-1(RNAi) was seen in worms treated with RNAi of genes against the β, B and O subunits. Thus, this effect of ATP-1 is independent of other ATP synthase subunits.

Since binding to ATP is a critical part of the role of the alpha subunit in ATP synthase, the inventors of the present invention sought to determine whether the ATP-binding domain is required for Ent interaction and in promoting the role of iron levels. Thus, the present inventors deleted residue 198-205(DRQTGKTA) from the sequence of the ATP-1 protein (FIG. 11D) and found that this deletion did not reduce the ability of the protein to bind to Ent as measured by in vitro binding assays (FIG. 4E), indicating that ATP-1-Ent binding is not associated with ATP-1-ATP binding. The inventors next tested whether transgenic expression of this ATP-1(del) protein was sufficient to function as ATP-1 in Ent-mediated iron uptake. As indicated in FIG. 4A, expression of this protein after the ribosomal gene promoter of the [ Prpl-28:: atp-1(del) ] transgene significantly inhibited the decrease in iron levels caused by the atp-1(lf) mutation. Thus, the function of ATP-1 in both interacting with Ent and promoting iron uptake is independent of its role in ATP binding.

Example 6: the enterobacterin-ATP-1 interaction promotes iron levels in host mitochondria.

Since iron transport into mitochondria greatly affects unstable iron pools and overall iron homeostasis, the present inventors sought to determine whether bacterial Ent and its interaction with host ATP-1 promoted iron levels in mitochondria. The inventors of the present invention first observed co-localization of ATP-1 and MitoTracker markers in the intestine (fig. 12A), consistent with ATP-1 function in mitochondria. The inventors then performed in vivo assays modified from protocols published for mammalian cells (deviredy et al, 2010) to examine the role of the Ent-ATP-1 interaction in promoting mitochondrial iron levels. Worms were treated with RNAi and fed 55FeCl₃+/-Ent, followed by mitochondrial isolation and radioactivity (55Fe) measurement. Mitochondrial iron (55Fe) increased three-fold with Ent supplementation, and this increase was dependent on ATP-1, but not on other ATP synthase subunits (fig. 5A). In addition, the inventors of the present invention showed that mitochondria isolated from worms fed with wild-type e.coli contained significantly higher levels of siderophore than worms fed with entF mutant bacteria, indicating that Ent also entered mitochondria (fig. 5B), and that when ATP-1 was reduced by RNAi, the level of Ent in mitochondria was significantly reduced (fig. 5C). Thus, bacterial Ent promotes increased host mitochondrial iron levels, and this process requires ATP-1 to have a novel function in the host mitochondria that is not associated with ATP synthase.

Example 7: ATP-1 plays a role in promoting increased levels of Ent-Fe3+ in mitochondria.

The ATP synthase alpha subunit resides in the mitochondrial matrix, and this protein is transported into the mitochondria by well-characterized mitochondrial protein transport mechanisms. Thus, ATP-1 can promote Ent-Fe through a "cotransport" model³⁺Input mitochondrial species, the co-transport model requires ATP-1 to bind to Ent prior to transport. To test this model, the inventors sought to determine whether synthesis or shuttling to ATP-1 could be performed in the absence thereofIn the case of mitochondria, the role of Ent and ATP-1 in purified mitochondria was observed.

In this in vitro assay modified from protocols published for mammalian cells (Devireddy et al, 2010; incorporated herein by reference), the inventors of the present invention first extracted mitochondria from RNAi-treated worms and then added 55FeCl₃+/-Ent, followed by 55Fe quantification. Addition of Ent increased iron (55Fe) levels in mitochondria by a factor of 10 greatly, and this increase was greatly reduced when the worms were treated with RNAi to ATP-1, but not for the worms treated with RNAi targeting the other three ATP synthase subunits (fig. 5D).

This result further demonstrates the role of Ent and ATP-1, but not the remainder of the ATP synthase, in promoting mitochondrial iron levels. Since it is unlikely that new ATP-1 protein will be produced in the assay mix, the results of this assay may exclude potential "co-transport" models and may indicate that ATP-1 facilitates mitochondrial Ent-Fe import by binding to Ent within mitochondria. This "retention" model further indicates that Ent-Fe³⁺Enter mitochondria by other means and may have a higher tendency to exit without ATP-1 interaction, which may be consistent with the assumption that Ent may enter mammalian cells by passive permeation. The stronger end effect observed in the in vitro assay (fig. 5D) compared to the in vivo assay (fig. 5A) may be due to the stronger affinity of end for the mitochondrial environment for solution under the in vitro assay conditions.

To observe the functional effects of end-mediated increases in mitochondrial iron levels, the inventors tested the effect of end on iron-dependent mitochondrial enzymes in helminths under the inventors' culture conditions. It was found that the activities of both mitochondrial Fe-S cluster enzymes aconitase and succinate dehydrogenase were significantly increased by supplementation with Ent (fig. 5E, F), demonstrating the effect of the Ent-ATP-1 complex in supplying iron to Fe-S clusters and other iron-containing molecules in mitochondria.

Example 8: ent interacts with ATP5a1 to promote mitochondrial iron uptake into mammalian cells.

Sequence ratioIt was shown that 78% identity exists between the alpha subunit of ATP synthase from C.elegans (ATP-1, nematode (Wormbase); SEQ ID NO.7) and human (ATP5A1, NCBI; SEQ ID NO.8) (FIG. 11D). Using CAS staining, the inventors of the present invention observed that Ent supplemented to the culture medium could enter human HEK293T cells (fig. 6A). To test whether this Ent-ATP-1 function was retained in mammals, the inventors of the present invention repeated the aforementioned biotin-Ent pull-down assay using total protein extracts of +/-biotin-Ent from human HEK293T cell culture (fig. 3A). ATP5a1 was clearly detected (fig. 6B), demonstrating that Ent also binds to ATP5a1 in mammalian cells. In an in vitro binding assay, biotin-Ent binds directly to the human protein ATP5a1, and the binding is effectively competed for by the presence of excess free Ent (fig. 6C). In a third assay, the inventors of the present invention added radiolabeled iron (55 FeCl) to cell lysate +/-Ent₃) Then IP was performed using ATP5a1 antibody. The presence of Ent increased the 55Fe level in the ATP5A1-IP sample (FIG. 6D). Together, these data indicate that the interaction between Ent and the a subunit of ATP synthase is retained in mammalian cells.

To test whether the function of the Ent-ATP-1 complex in promoting mitochondrial iron uptake was also retained in human cells, the inventors of the present invention performed both in vivo and in vitro mitochondrial iron uptake assays and found that the addition of Ent significantly increased the iron level in mitochondria in both assays (fig. 6E, F). Furthermore, using siRNA knockdown (fig. 13A), the inventors observed that this Ent-mediated increase was dependent on ATP5a1 (fig. 6E, F) in a similar manner to the assay against c.elegans (fig. 5A, D). The inventors also measured mitochondrial iron levels in cells by using the fluorescent mitochondrial iron indicator RPA, the binding of iron to RPA quenching the fluorescence of RPA. Supplementation with Ent increased mitochondrial iron levels in cells, and this iron-elevating effect was not seen in cells treated with ATP5a1 sirnas (fig. 6G). The relatively minor changes seen for the cultured cells compared to the differences in the worms fed heat-inactivated food (fig. 2) may potentially be due to higher levels of pro-iron under cell culture conditions. These data demonstrate that Ent affects mammalian mitochondrial iron levels and in an ATP5a 1-dependent manner.

Example 9: in media with iron chelators, end addition increased iron uptake in human HEK293 cells.

In fig. 2G, the inventors of the present invention show the strong effect of Ent to move iron into live c. Here, a similar test was performed in human HEK293 cells. When the iron chelator Desferrioxamine (DFO) was added to the medium, the iron levels in the cells were significantly reduced as indicated by an increase in calcein AM staining fluorescence. By adding Ent (1.5 μ M) to the medium, the iron level was substantially restored. The decrease in calcein AM fluorescence with the addition of Ent (45%) was stronger than the test without DFO as shown in fig. 6G, indicating that the low iron condition was more sensitive to the presence of Ent. This result demonstrates the effectiveness of Ent to move iron (low levels of free iron or iron bound to DFO) into cells.

Example 10: ent and ATPS α promote iron transport across the lipid bilayer of the liposome.

The inventors used synthetic liposomes in vitro to test the ability of Ent to move iron across lipid bilayers. Briefly, FeCl is added₃+/-Ent (1.5. mu.M) was added to fresh liposomes formed from commercial Lipids (Avanti Lipids). ATPS α (ATP-1 or ATP5a1) was added to the liposomes by established methods. Fe associated with liposomes, as shown in FIG. 19³⁺Level of

Measured by two methods: (A) calcein AM staining: calcein AM dye was added to liposomes +/-ATPS α and the fluorescence intensity of the liposomes was measured; (B) combining liposomes with radiolabeled Fe³⁺(⁵⁵FeCl₃) Liposomes were incubated and measured for radioactivity (relative CPM). Since calcein AM is present only inside, method (a) measures iron inside the liposomes. Method (B) is a simpler method that may not exclude iron outside the liposomes. In each test, the presence of Ent significantly elevated the iron inside the liposomes (a) or associated with the liposomes (B)And (4) horizontal.

Example 11: ent supplementation by oral gavage promoted hemoglobin in anemic mouse model (dietary anemia) And increased spleen iron levels.

The inventors fed an Iron Deficiency Diet (IDD) (or control diet) to 3-week-old female mice for 6 weeks to induce anemia (confirmed by hemoglobin measurements). Then +/-Ent (two concentrations) or +/-FeS0 by oral gavage (once every two days) over a two week period₄Mice (5/group) were treated. As shown generally in fig. 20A-B, feeding IDD caused a substantial decrease in both hemoglobin and iron levels. Despite the large error bars, end supplementation partially but significantly restored hemoglobin levels, suggesting that end may have an effect on increasing iron uptake efficiency under severe anemia conditions. With the addition of Ent, iron levels in the spleen increased, but iron levels in the liver did not. Hemoglobin levels were obtained using a commercial kit (BioAssay system). Under normal conditions, about 70% of the iron absorbed from the intestine is used to produce hemoglobin, and if there is an excess of iron, only a small percentage can be stored in the liver or other somatic tissues. Under anemic conditions, a large percentage of the iron that enters the mitochondria (and then binds to heme) can be transported to erythropoiesis. Therefore, it is not surprising to see that end supplementation has little effect on liver iron levels (iron stores).

Example 12: ent-facilitated hemoglobin in anemic mouse model (dietary anemia) supplemented by drinking water (ad libitum) The level increased.

The inventors fed an Iron Deficiency Diet (IDD) (or control diet) 5 weeks to 3-week-old male mice to induce anemia. The mice were then fed IDD +/-Ent added to drinking water for another two weeks. Fresh dilutions of Ent in water were provided once a week. As shown generally in fig. 21, mice continuously fed an iron-replete Control Diet (CD) were included as a control. Hemoglobin levels were measured by a Hemavet hematology analyzer. Mean ± SD are shown. End supplementation significantly increased hemoglobin levels in anemic mice. Since it was later found that Ent is highly unstable in the deionized water used in the test (pH 5.5) (see fig. 25), the effect of Ent may be reduced in this test. Future tests will administer Ent in pH adjusted water and will contain more frequent dilutions of fresh Ent to ensure stability.

Example 13: ent supplemented by drinking water (ad libitum) promoted the growth of mice fed a control (iron-replete) diet.

The inventors of the present invention treated male mice for 4.5 weeks with the above-described control iron-rich Control Diet (CD) matched to the iron-deficiency diet (IDD). As shown above in fig. 20 and 21, mice fed this diet had relatively normal hemoglobin and iron levels. As further shown in figure 22, mice had increased growth rates when Ent was supplemented in drinking water (pH 5.5). This laboratory is incomplete because hemoglobin levels and iron levels are not measured, and this will be repeated. However, the weight gain data was still significant, as the inventors have shown that Ent has a profound effect on larval growth of c.elegans, and that the effect is due to the ability to promote an increase in the iron level of the animals (figures 1 and 2).

Example 14: ent promotes the growth of mice colonized with a single strain of e.

As shown generally in fig. 23, (a) five-week-old female sterile (GF) mice were colonized with a single non-pathogenic e.coli (K12) strain, wild-type or entF (e.coli lacking Ent). Mouse growth (weight gain) was measured over the next 4 weeks. Sterile (GF) mice colonized with entF e. Interestingly, the difference in weight gain was greatest during the first two weeks after colonization. (B and C) iron levels in late mouse were only significantly lower in spleen (about 35%) but not in liver or other tissues, consistent with the results seen in fig. 20. The iron level was measured. (D) The Ent supplementation overcomes the growth delay of GF mice colonized with entF (-) e. GF female mice colonized with entF bacteria were supplemented with Ent [2 concentrations were added to drinking water (pH 5.5) once weekly ].

The data indicated significant recovery of growth. The Ent effect was more pronounced during the first 2 weeks. It may be noted that the effect of Ent here may be limited due to its instability in low pH water. Additional embodiments may administer Ent in pH adjusted water and will further contain more frequent dilutions of fresh Ent to ensure stability. Since Ent is used by e.coli to support its growth, colonization in the gut may be reduced in the absence of Ent, and the developmental delay within the first 2 weeks may be due to the indirect effect of less e.coli in the gut. However, such large weights are unlikely to be due to potential differences in E.coli colonization. More importantly, the inventors have shown that the role of Ent in promoting caenorhabditis elegans development is not related to the bacterial use of Ent (see fig. 1F) and that Ent biosynthesis has no significant effect on intestinal colonization by caenorhabditis elegans (Qi and Han, 2018). The difference in weight gain between 1-2 weeks and 3-4 weeks may indicate that Ent (or e.coli) has a more prominent effect in the early stages, where faster growth requires more iron acquisition. Alternatively, over the next two weeks, the mice may have some adaptive/compensatory changes (less dependent on Ent).

Example 15: the effect of enterobactin (Ent) in promoting animal development was not seen in other siderophores.

After the inventors' established assays (Qi and Han,2018), newly hatched caenorhabditis elegans larvae were fed either wild-type K12 e.coli or entF e.coli supplemented with the indicated siderophores as shown in figure 24. In this assay, Ent is necessary to support the growth of caenorhabditis elegans. The volume of worms was measured after 3 days (as performed for the experiment described in figure 1). While supplementation with enterobactin restored the growth defect seen with entF, the other five siderophores tested failed to show an effect (consistent with the other results in fig. 1G). Thus, the role of Ent in promoting iron transport and animal development is specific to Ent.

Example 16: materials and methods.

BeautifulCryptocorynebacterium sp.Nematode stocks were maintained at 20 ℃ on Nematode Growth Medium (NGM) plates inoculated with bacteria (e.coli OP 50). The following strains/alleles were obtained from the Caenorhabditis Genetics Center (CGC): n2 Bristol (known as wild type); VC2824: H28Ol6.l (ok2203) I/hT2[ bli-4(e937) let-? (q782) qls48](I；III)；XA6901:qaEx690l[ftn-2p::pes-lO::GFP::his+lin-l 5(+)]；SJ4l03:zclsl4[myo-3::GFP(mit)]. For the Prpl-28: ATP-1(del) transgene, the ATP-binding sequence (residue 198-.

A cell line.HEK293T cells were obtained from ATCC and maintained in humidified cabinet with 5% CO2 at 37 ℃. Cells were cultured with DMEM supplemented with 10% FBS, 4mM L-glutamine, 100 units penicillin/mL, 100. mu.g streptomycin/mL, and 0.25. mu.g amphotericin B/mL.

And E.coli Keio collection and screening.Heat-inactivated (HK) OP50 plates were prepared after the preceding procedure (Qi et al, 2017). A standard overnight culture of E.coli OP50 grown in LB broth was concentrated to 1/10 volume and then heat inactivated in a 75 ℃ water bath for 90 minutes. 150 μ l of HK-OP50 was spread on one side of the NGM plate. For preparation of bacterial mutant assay plates, E.coli Keio (Baba et al, 2006) mutants were grown overnight at 37 ℃ in LB medium with 10mg/mL kanamycin (kanamycin). 0.2. mu.L of bacterial culture (OD)₆₀₀) Seeded on the other side of the HK OP50 plate. Approximately 300 synchronized L1 worms were added to the screening plates and cultured at 20 ℃ before grading the worm size on days 3 and 4. The whole library is used for primary screening; each bacterial mutant was screened once. For the secondary screening, 200 candidate mutants (3 replicates) were screened to confirm the slow-growing phenotype.

Chemical supplementation of the plates.For chemical supplementation, each chemical was dissolved in water or DMSO to generate stock solutions. Stock solution was added to HK OP50 andand then spotted onto the NGM board. The chemical name, supplier, stock solution concentration and volume for each chemical are listed below: 2,3-DHBA (Sigma 126209-5G, 300mM, 5. mu.l), enterobactin (Sigma E3910-1MG, 1MG/ml, 5. mu.l), fluorescein (Sigma P8124-1MG, 0.5MG/ml, 5. mu.l), ferrichrome (Sigma F8014-1MG, 0.5MG/ml, 5. mu.l), heme (Sigma 51280, 1MG/ml, 5. mu.l), FeCl3 (Sigma 236489, 175. mu.g/. mu.l, volume indicated in the assay). For the CaEDTA supplementation (Klang et al, 2014), 50. mu.l of CaEDTA (50. mu.g/. mu.l) were spread in the center of an OP50 seeded NGM plate, and then different volumes of FeCl3[ 175. mu.g/. mu.l ]]Ent (0. mu.l, 1. mu.l, 5. mu.l, 10. mu.l, 50. mu.l) or 20. mu.l (0.5mg/ml) was added to the center of the lawn.

And (5) analyzing the bacterial growth.The overnight culture was diluted to final OD in 200. mu.L of NGM liquid medium₆₀₀0.01. Bacteria were grown in 96-well plates for 17 hours at 37 ℃ by shaking in a Synergy2 plate reader (BioTek). OD was recorded at 20-minute intervals₆₀₀。

Bacterial colonization in a helminth assay.Bacterial colonization by caenorhabditis elegans was determined using a method adapted from published procedures (Portal-Celhay and blast, 2012). Briefly, stage L3 worms were collected from NGM plates and washed 3 times over a large area with 10mL of M9 buffer. The animals were then placed in empty NGM plates with 100mg/mL ampicillin (ampicillin) for 1 hour to remove surface bacteria. 10 worms were individually picked up in M9 buffer and homogenized by sonication. Part or all of the mixture was then spread onto LB plates. After incubation at 37 ℃ overnight, the number of bacterial colonies was determined.

Quantification of siderophore.CAS agar plates were prepared according to published methods (Schwyn and Neilands, 1987). The worms fed wild-type or entF mutant e.coli were then collected, washed 5 times with 10mL M9, and then starved overnight in 10mL M9 to digest and clear the enteric bacteria. The worms were then homogenized by sonication and measured by BCA protein assay kit (thermo fisher, 23225)Protein concentration of supernatant. Protein input was normalized based on protein concentration. The supernatant was placed on CAS agar plates and incubated overnight at room temperature and monitored for the formation of an orange halo and the color intensity was quantified by image J. CAS exhibited a unique blue color when complexed with iron. When iron is sequestered by the siderophore, an orange halo forms around the sample. The intensity of halo formation is directly proportional to the concentration of the siderophore.

Analysis of larval growth by measuring worm size.The synchronized L1 worms were seeded on the indicated NGM plates and grown to the indicated times. Photographs were taken and the volume of worms in each photograph was measured by WormSizer software (Moore et al, 2013).

Iron was determined in the worms.As previously described, iron in worms was imaged in real time (James et al, 2015). Briefly, worms were collected under different culture conditions. Then co-cultured in M9 with 0.05. mu.g/ML calcein-AM (Invitrogen) for 1 hour, followed by 3 washes in 1ML M9. The samples were then mounted for fluorescence microscopy.

Western blotting.To measure the level of ATP synthase α subunit, either the cells treated with RNAi or siRNA were analyzed by standard western blotting and probed with anti-ATP synthase α subunit (dilution l: 5000; sefmeishel, 43-9800) and anti-actin (dilution l: 5000; sigma-a 2066) as loading controls.

Isolation of enterobacterin-binding proteins by biotin-IP and LC-MS.Total protein was extracted from mixed stage worms and then by adding 100. mu.l

M-280 streptavidin was pre-washed three times. Equal volumes of these total protein extracts were then divided into two tubes. Biotin-Ent (5. mu.g) was added to one tube for IP and biotin alone (5. mu.g) was added to the other tube as a control, both incubated overnight at 4 ℃. And

after 2 hours incubation with M-280 streptavidin, the beads were washed at least 3 times by 1mL PBS. PBS was then removed from the beads and 200. mu.L of 0.1M Ammonium Bicarbonate (ABC)/0.00L% deoxycholic acid (DCA) was added. The samples were reduced using 5mM (final) TCEP at 60 ℃ for 30 minutes and alkylated using 15mM iodoacetamide at room temperature for 20 minutes. 0.5. mu.g of trypsin was added to each sample and incubated overnight. The sample was then acidified using 7uL of formic acid. DCA was removed from the sample by phase transfer using ethyl acetate. Samples were desalted using a Pierce C18 spin column and dried using a speed vac. Samples were reconstituted in 10. mu.L of buffer A (0.1% formic acid in water) and 5. mu.L of this was analyzed by LC-MSMS.

Determination of the interaction of Enterobacteriaceae element and ATP-1 protein. In vivo binding assay: the worms were grown with an end-biotin (5 μ g/ml) dietary supplement and then subjected to streptavidin bead IP. Western blotting was performed using an antibody against mammalian ATP synthase alpha subunit (Saimer Feishel Co., 43-9800) to detect ATP-1.

In vitro binding assays.(a) End-biotin pull-down of total protein: interactive proteins from total worm protein extracts were pulled down using Ent-biotin and streptavidin beads (same method as in the preliminary screen for Ent-binding proteins). Western blotting was performed to detect ATP-1 (Saimer Feishel Co., 43-9800). (b) Binding of Ent-Biotin to purified ATP-1:: HIS-labeled protein in assay buffer (50mM 2- [4- (2-hydroxyethyl) -l-piperazinyl) at 30 ℃]Ethanesulfonic acid (HEPES) pH 8.0, 100mM NaCl, 0.5mM Dithiothreitol (DTT)) was purified with Ent-biotin (1 μ g/μ L; the purified protein was treated with 1mg/ml stock of) +/-Ent (1. mu.g/. mu.l) in DMSO for 1 hour for a total assay volume of 20. mu.L. The assay was then quenched (and thus reduced) with standard 5X SDS-PAGE loading buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking the membranes in 5% BSA Tris Buffered Saline (TBS) with 0.1% Tween 20(TBST) for 1 hour at room temperature, followed by horseradish peroxidase streptavidin (Cell Signaling technology, Inc. ()Signaling Technology), 3999S) were incubated in TBST for 1 hour. After four washes in TBST with changes every 15 min, biotinylated proteins were visualized by enhanced chemiluminescence (GE Healthcare, RPN 2232). When the binding to Ent was at the maximum level of 1/2, Ka was calculated as the concentration of ATP-1-His protein. (c) Ent dependence for ATP-1 interaction with iron: radiolabelled iron (55 FeCl)₃1uCi) or 55FeCl₃(1uCi) + Ent (2. mu.g) was added to the worm lysates and then immunoprecipitated using an antibody against ATP-1 (dilution ═ l: 5000; Sammel Feishel, 439800). After IP, the amount of 55Fe was determined by liquid scintillation.

Immunofluorescence.Antibody staining was performed as described previously (Zhang et al, 2007). Briefly, L1 wild-type worms (N2) were treated by feeding atp-1RNAi and grown to the L4 stage. Before antibody staining, worms were grown for 1 day at 20 ℃ on NGM agar supplemented with 1. mu.g/ml MitoTracker Red (cell signaling technology Co., Ltd. # 9082). Dissected worms were fixed in 3% formaldehyde with 6mM K2HPO4 (pH 7.2) and 75% methanol for 10 minutes at-20 ℃. The fixed worms were rinsed three times in PBS and blocked for 1 hour at room temperature in PBS containing 0.5% BSA and 0.1% Tween-20. anti-ATP synthase alpha subunit (diluted 1: 200) and anti-rabbit antibody (diluted 1: 400) (Invitrogen, A11011) were used as primary and secondary antibodies, respectively.

And (3) RNAi treatment.L1 worms were processed and grown to adults by feeding first generation RNAi (Ahringer, "Reverse genetics") 2006. Adults were then bleached and allowed to incubate in M9 buffer for 18 hours. Synchronized L1 worms were seeded on heat-inactivated OP50 plates with entF bacteria or heat-inactivated OP50 plates supplemented with Ent. After 4 days, worm size was measured. To determine the effect of the different ATP synthase subunits on the iron levels of the worms, L1 worms were subjected to RNAi feeding and grown to young adults. The iron level of the worms at the same stage was measured. L1 helminths were treated and grown for in vitro/in vivo mitochondrial iron uptake with RNAi targeting the indicated ATP synthase subunitTo young adults and then subjected to further procedures.

siRNA treatment in mammalian cells.ATP5a1 siRNA (SASI _ Hs01_00119735) was purchased from sigma. According to the manufacturer's instructions, use

RNAiMAX transfection reagent (seimer feishel, 13778075) delivered siRNA into HEK293T cells. Knockout efficiency was assessed by immunoblotting.

Mitochondrial iron uptake assay.The inventors of the present invention modified the published procedures for analyzing mammalian cells for in vitro mitochondrial iron uptake assays (deviredy et al, 2010). Specifically, 1uCi 55FeCl was applied at room temperature₃Incubate with 2 μ g iron-free Ent (1mg/ml in DMSO) or DMSO for 3 hours before adding mitochondria purified from either worms treated with different RNAi or cells treated with siRNA. The samples were incubated at room temperature for 4 hours and the amount of 55Fe in the lysed mitochondria was determined by liquid scintillation.

Mitochondrial iron uptake assays in vivo were also improved based on published procedures (Devireddy et al, 2010). Specifically, 1uCi 55FeCl was applied at room temperature₃Incubate with 2. mu.g iron-free Ent (1mg/ml stock in DMSO) or DMSO for 3 hours. Addition of 55FeCl to juvenile adults treated with first generation RNAi₃+ DMSO or 55FeCl ₃ + enterobacterin.

After overnight growth, the worms were washed in M9. Then, mitochondria were separated, and then the amount of incorporated 55Fe was measured by a liquid scintillation method.

Mitochondrial iron measurement in mammalian cells.Mitochondrial iron pools were determined as described (Mena et al, 2015). Briefly, 2. mu.M of the mitochondrial iron chelator rhodamine B- [ (1, 10-phenanthroline-5-yl) aminocarbonyl]Benzyl ester (RPA) cells were loaded for 20 min at 37 ℃. After washing, the cells were imaged by fluorescence microscopy.

And (3) extracting mitochondria.Mitochondrial isolation kit (Mito) for use with cultured cellsMitochondria were extracted from HEK293T Cells using the chondria Isolation Kit for filtered Cells (seimer feishel, 89874). Mitochondria were extracted from worms using a Mitochondria Isolation Kit (Mitochondria Isolation Kit for Tissue) (seimer feishel corporation, 89801).

An enzyme activity.The enzyme activities of succinate dehydrogenase (MAK 197; Sigma) and cis-aconitase (MAK 051; Sigma) were measured using the kit according to the manufacturer's protocol. Briefly, the L1 worms were seeded on the assay plates +/-Ent. After 48 hours incubation, worms were lysed under ice-cold conditions using lysis buffer provided in the kit supplemented with protease. Equal amounts of protein were used for enzyme activity assays.

And (5) carrying out microscopic examination.Fluorescence analysis was performed on a Zeiss Axioplan2 microscope with a Zeiss AxioCam MRm CCD camera under Nomarski optics. Plate phenotype was observed using a Leica MZ16F dissecting microscope with a Hamamatsu C4742-95 CCD camera.

And (4) quantifying.ImageJ software was used for quantification of calcein-AM staining and western blotting for the Ent-protein binding assay. For calcein-AM staining, raw images taken with the GFP channel were used to measure intensity. Staining intensity values were determined by subtracting background intensity from calcein-AM stained intestine.

And (5) carrying out statistical analysis.Except by using χ²Out of FIGS. 1I, 2G and 11B of test analysis, all statistical analyses were performed using student's t-test, and p was assigned<0.05 was considered a significant difference.

Watch (A)

Table 1. potential Ent binding proteins identified by mass spectrometry.

Reference to the literature

The following references are hereby incorporated by reference in their entirety:

[1] baba, t., Ara, t., Hasegawa, m., Takai, y., Okumura, y., Baba, m., Datsenko, k.a., Tomita, m., Wanner, b.l., and Mori, H. (2006) construction of an in-frame single gene knockout mutant of e.coli K-12: molecular systems biology (Mol Systbiol) 2,20060008, Keio Collection (Construction of Escherichia coli K-12in-frame, single-gene knockout variants).

[2] Barrat, m.j., Lebrilla, c., shariro, h.y, and Gordon, J.I, (2017) gut microbiota, food science, and human nutrition: cell Host microorganism (Cell Host microorganism) 22,134-141, was also incorporated at The appropriate time (The Gut microbial, Food Science, and Human Nutrition: A time Marriage). Baumler, a.j. and spectandio, v. (2016.) Interactions between microorganisms and pathogenic bacteria in the gut (Interactions between the microbial and pathogenic bacteria in the gut), Nature 535, 85-93.

[3] Berg, M., Stenuit, B., Ho, T, Wang, A., Parke, C., Knight, M., Alvarez-Cohen, L., and Shapira, M. (2016.) the Assembly of the gut microbiota of C.caenorhabditis elegans from different soil microbial environments (Assembly of the Caenorhabditis elegans microorganisms from microbial soil microorganisms) & ISME J10, 1998-.

[4] Blaton, l.v., barrat, m.j., Charbonneau, m.r., Ahmed, t, and Gordon, J.I, (2016) (Childhood undersuity, the gut microbiota, and microbiota-directed therapeutics) Childhood university Science (Science) 352,1533.

[5] Brown, j.m. and Hazen, s.l. (2018.) Microbial modulation of cardiovascular disease. nature comments: microbiology (Nat Rev Microbiol) 16, 171-.

[6] Cabreiro, F., Au, C., Leung, K.Y., Vergara-Irigarey, N., Cocheme, H.M., Noori, T., Weinkov, D., Schuster, E., Greene, N.D., and Gems, D. (2013). Methylbiguanide retards senescence of C.elegans by altering the metabolism of microbial folate and methionine (for metabolic recovery in C.elegans by alteration of microbial folate and methionine) Cell (Cell) 239.

[7] Cassat, J.E. and Skaar, E.P. (2013.) Iron in infection and immunization [ cell host microorganisms ] 13,509 and 519.

[8] Charbonneau, m.r., O' Donnell, d., blaton, l.v., Totten, s.m., Davis, j.c., barrat, m.j., Cheng, j., Guruge, j., Talcott, m., Bain, j.r. et al (2016.) Sialylated Milk Oligosaccharides Promote microorganism-Dependent Growth in Infant malnutrition Models (sialated Milk Oligosaccharides) cell 164, 859-.

[9] The Diversity of structures and properties of catalases (Diversity of structures and properties of catalases), Cell and molecular Life sciences (Cell Mol Life Sci.) 61,192-208 has been reported. Deverieddy, L.R., Gazin, C., Zhu, X, and Green, M.R (2005.) cell surface receptors for lipoproteins 24p3 selectively mediate apoptosis and iron uptake (A cell-surface receptor for lipid 24p3 selective mediators apoptosis and iron uptake.). cell 123, 1293-cell 1305.

[10] Deverieddy, L.R., Hart, D.O., Goetz, D.H., and Green, M.R (2010). A mammalian siderophore synthesized by enzymes and bacterial homologues involved in the production of enterobacterin (A mammalian siderophore synthesized by an enzyme with a bacterial homolog in enterobacterin production). cell 141, 1006-.

[11] Dirksen, p., Marsh, s.a., Braker, i., Heitland, n., Wagner, s., Nakad, r., Mader, s., Petersen, c., Kowallik, v., rosenstel, p, et al, (2016) the natural microbiota of the nematode caenorhabditis elegans: a pathway to a new host-microbiome model (The native microbiome of The microbial harbor organisms: gateway to a new host-microbiome model), BMC Biol 14, 38.

[12] Donia, m.s. and Fischbach, M.A. (2015) human microbiota: small molecules from the HUMAN microbiota (HUMAN microbiota) science 349,1254766.

[13] Ellermann, M. and Arthur, J.C, (2017) Siderophore-mediated iron acquisition and modulation of host-bacterial interactions (Free-mediated iron acquisition and modulation of host-bacterial interactions 105,68-78 Free radical biology and medicine (Free radial Biol Med).

[14] Flo, t.h., Smith, k.d., Sato, s., Rodriguez, d.j., Holmes, m.a., Strong, r.k., Akira, s, and Aderem, a. (2004) Lipocalin 2mediates an innate immune response to bacterial infection by chelating iron (Lipocalin 2 peptides an immune response to bacterial infection by sequencing) & nature 432, 917-.

[15] Ford, s.a., Kao, d., Williams, d, and King, K.C, (2016) microorganism-mediated host defense drives the evolution of reduced pathogen virulence (Microbe-mediated host infection drive of reduced pathogen virus): nature communication (Nat command) 7,13430.

[16] Froisnard, M., Belgareh-Touze, N., Dias, M., Buisson, N, Camadr, J.M., Haguenauer-Tsias, R, and Lesuise, E. (2007). transportation of siderophore transporters and intracellular fate of sidechain B conjugates in Saccharomyces cerevisiae [ transportation (transactional of siderophore transporters in Saccharomyces cerevisiae and intracellular fate of ferredox B conjugates ] (Transfftic) 8, 1601-.

[17] Garcia-Gonzalez, A.P., Ritter, A.D., Shrestha, S., Andersen, E.C., Yilmaz, L.S. and Walhout, A.J.M. (2017). Bacterial Metabolism Affects the C.elegans Response to Cancer chemotherapy (Bacterial Metabolism effects to Cancer Chemotherapeutics). Thermothers 169,431-441e 438.

[18] Grillo, a.s., santamaia, a.m., Karina, m.d., Cioffi, a.g., Huston, n.c., Han, m., Seo, y.a., Yien, y.y., Nardone, c., Menon, a.v., et al, (2017) iron transport Restored by small molecules promotes absorption and hemoglobin proteolysis in animals (Restored iron transport by a small molecule) scientific 356,608-one 616.

[19] Gusarov, I., Gautier, L., Smolensteva, O., Shamovsky, T, Eremina, S., Mironov, A., and Nudler, E. (2013). Bacterial nitric oxide extends the life of C.elegans (Bacterial nitrile oxide extensions of the life span of C.elegans.). cell 152,818- & 830.

[20] Han, B., Sivaramakrishnan, P., Lin, C.J., Neve, I.A.A., He, T, Tay, L.W.R., Sowa, J.N., Sizovs, A., Du, G., Wang, T et al (2017). the Genetic Composition of microorganisms fine-Tunes the Longevity of the Host (Microbial Genetic compositions tasks Host Longevity).: cell 169,1249-1262 l 3.

[21] Hider, R.C. and Kong, X. (2010) Chemistry and biology of siderophores (Chemistry and biology of siderophores), Natural products report (Nat Prod Rep) 27, 637-657.

[22] James, s.a., Roberts, b.r., Hare, d.j., de change, m.d., bircell, i.e., Jenkins, n.l., Cherny, r.a., Bush, a.i., and McColl, g. (2015) Direct in vivo imaging of ferrous iron dysom formation in Caenorhabditis elegans for imbalance in ferrous homeostasis of senescent cryptorhabditis elegans (Chem Sci) 6,2952 + 2962.

[23] Johnson, d.c., Dean, d.r., Smith, a.d., and Johnson, M.K (2005.) Structure, function, and formation of biological iron-sulfur clusters (Structure, function, and formation of biological iron-sulfur clusters) & annual review of biochemistry (Annu Rev Biochem) 74, 247-.

[14] Junge, W. and Nelson, N. (2015.) ATP synthase (ATP synthase.) "annual review of biochemistry" 84, 631-657. Kakhlon, o. and Cabantchik, Z.I. (2002). unstable iron bath: characterization, measurement and participation in cellular processes (l) (The laboratory ion: chromatography, measurement, and differentiation in cellular processes (l)). 33, 1037-.

[15] Kirienko, N.V., Ausubel, F.M., and Ruvkun, G. (2015) resistance to siderophore-mediated killing by Pseudomonas aeruginosa by mitochondrial autophagy.Proc. Natl Acad Sci U S.A. App.772, 1821 and 1826.

[16] Kirienko, N.V., Kirienko, D.R., Larkins-Ford, J., Wahlby, C., Ruvkun, G., and Ausubel, F.M, (2013). Pseudomonas aeruginosa disrupts iron homeostasis of C.elegans, causing hypoxic reactions and death (Pseudomonas aeruginosa strains of Caenorhabditis elegans hormone and death) & cell host microorganism & 13,406. 416.

[17] Klang, i.m., Schilling, b., Sorensen, d.j., Sahu, a.k., Kapahi, p., Andersen, j.k., Swoboda, p., killelea, d.w., Gibson, b.w., and Lithgow, G.J, (2014) Iron promotes protein insolubility and Aging of cryptorhabdus elegans (Iron protein insolubility and Aging c.elegans) & Aging (Aging) (Albany NY) 6,975- & 991.

[18] Koppel, N. and Balskus, E.P, (2016.) exploration and understanding of the Biochemical Diversity of the Human Microbiota (expanding and expressing the Biochemical Diversity of the Human Microbiota), cytochemical biology (Cell Chem Biol) 23, 18-30.

[19] Kortman, g.a., Dutilh, b.e., Maathuis, a.j., Engelke, et.f., Boekhorst, j., Keegan, k.p., Nielsen, f.g., Betley, j., Weir, j.c., Kingsbury, z. et al (2015). In an In vitro Model of TIM-2 of the Human Colon, Microbial Metabolism Shifts toward an adverse condition with supplemental Iron (Microbial Metabolism approaches and additives with complementary particles In the TIM-2In vitro Model of the Human Colon) & lttm microbiology Front 6,1481.

[20] Kundu, p., Blacher, e., Elinav, e, and Pettersson, s. (2017). The Evolving intrinsic Self (Our Gut micro: The evaporating Inner set Self), cell 777, 1481-1493.

[21] Kwon, o., Hudspeth, m.e., and Meganathan, r. (1996) anaerobic biosynthesis of enterobactin e.coli: journal of bacteriology (J Bacteriol) 178,3252 and 3259, evidence for the regulation of the expression of the entC gene and its involvement in the biosynthesis of menaquinone (vitamin K2) (antibacterial biosynthesis of enterobacter Escherichia coli: regulation of the expression of entC gene and evidence for the inhibition of expression of the enzyme in the biosynthesis of menaquinone (vitamin K2)).

[22] Leunier, f., MacNeil, l.t., Lee, w.j., Rawls, j.f., canti, p.d., Schwarzer, m., Zhao, l., and Simpson, S.J. (2017). Cell metabolism (Cell Metab) 25,522-534 in Nutrition, Microbiota, Animal Physiology and "Crossroads" for Human Health.

[23] Liu, L, Rutz, J.M., Feix, J.B., and Klebba, P.E, (1993) Permeability properties of major gated channels within the FepA iron enterobacterin receptor (FepA) Properties of a large gated channel with the said magnetic energy receptor (FepA). Proc. Natl.Acad.Sci.USA 90, 10653-.

[24] Lloyd-Price, L, Mahurkar, A., Rahnavard, G., Crabtree, L, Orvis, L, Hall, A.B., Brady, A., Creasy, H.H., McCracken, C., Giglio, M.G., et al (2017), expanded Human Microbiome Project, Nature 550, 61-66.

[25] Meisel, j.d., Panda, o., mahanni, p., Schroeder, f.c., and Kim, D.H (2014.) chemosensory modulation of bacterial secondary metabolites regulates neuroendocrine signaling and behavior of c.elegans (chemosensory of bacterial secondary metabolites neuroendocrine signaling and biochemical of c.elegans) 159, 267-280.

[26] Mena, N.P., Garcia-Beltran, O., Lourido, F., Urrutia, P.J., Mena, R., Castro-Castillo, V., Cassels, B.K., and Nunez, M.T, (2015) novel mitochondrial iron chelator 5- ((methylamino) methyl) -8-hydroxyquinoline prevents mitochondrial-induced oxidative damage and neuronal death (Biochem Biophys Res Commun.) (463, 787-) 792.

[27] Moore, b.t., Jordan, j.m., and Baugh, L.R, (2013) WormSizer: high throughput analysis of nematode size and shape (Wormsizer: high-throughput analysis of nematode size and shape) public science library: general (PLoS One) 8, e57l 42.

[28] Muckenthaler, m.u., rivela, s., hentz, m.w., and Galy, b. (2017). Red Carpet for Iron Metabolism (a Red Carpet for Iron Metabolism), cell 168, 344-.

[29] Portal-Celhay, C. and Blaser, M.J. (2012.) Competition and resilience between the founder in the C.elegans gut and the introduced bacteria (Competition and resilience between the founder and the introduced bacteria in the C.elegans section gut.) infection and immunization (infection Immun) 80, 1288-1299.

[30] Postler, t.s. and Ghosh, s. (2017) understanding symbiotic functions: how Microbial Metabolites Affect Human Health and Shape the Immune System (the: How Microbial Metabolites Affect Human Health and Shape the Immune System), cell metabolism 26, 110-.

[31] Qi, B, Kniazeva, M, and Han, m. (2017) vitamin B2-responsive mechanisms that regulate intestinal protease activity to affect the dietary behavior and growth of animals (a vitamin-B2-sensing mechanisms that regulate enzyme activity to effect animals' food life and growth) & E life (eife) 6.

[32] Rangan, K.J., Pedicord, V.A., Wang, Y.C., Kim, B., Lu, Y, Shaham, S., Mucida, D, and Hang, H.C. (2016.) secreted bacterial peptidoglycan hydrolases enhance tolerance to enteric pathogens (A secreted bacterial peptidoglycan hydrolases) science 353, 1434-.

[33] Rauen, u., Springer, a., Weisheit, d., Petrat, f., Korth, h.g., de Groot, h., and Sustmann, R. (2007). chelatable mitochondrial iron was evaluated by using mitochondrial-selective fluorescent iron indicators with different iron binding affinities (association of chemical binding iron by using mitochondon-selective fluorescent iron indicators) with biochemical iron-binding definitions 8,341 flation 352.

[34] Raymond, k.n., Dertz, e.a., and Kim, s.s. (2003). Microorganism iron transport prototypes (Enterobacterin: an biochem for microbial iron transport.). Proc. Natl. Acad. Sci. USA 100, 3584-3588.

[35] Romney, s.j., Newman, b.s., Thacker, c, and Leibold, e.a. (2011) HIF-l regulates iron homeostasis of c.elegans by activating and inhibiting genes involved in iron uptake and storage (HIF-l regulation of iron homeostasis in Caenorhabditis elegans by activation and inhibition of genes involved in iron uptake and storage.) "public science library: genetics (PLoS Genet) 7, el 002394.

[36] Saha, p., Yeoh, b.s., Olvera, r.a., Xiao, x, Singh, v., Awasthi, d., Subramanian, b.c., Chen, q., Dikshit, m., Wang, y, et al (2017), Bacterial Siderophores hijacking neutrophiles Functions (2017), journal of immunology (J Immunol) 198, 4293-.

[37] Samuel, b.s., rowelder, h., braidlet, c., Felix, m.a., and Ruvkun, g. (2016.) reaction of c.elegans to bacteria from its natural habitat (Caenorhabditis elegans et al, journal of the national academy of sciences, 113, E3941-3949.

[38] Schwyn, B. and Neilands, J.B (1987). general chemical assays for the detection and determination of siderophores 160,47-56, analytical biochemistry (Anal Biochem).

[40] Scott, T.A., Quinteneio, L.M., Norvasias, P., Lui, P.P., Wilson, M.P., Leung, K.Y., Herrera-Dominguez, L., Sudiwala, S., Pessia, A., Clayton, P.T., et al (2017). Co-metabolism of Host-microorganisms determines the Efficacy of Cancer drugs in C.elegans (Host-Microbe microorganisms Cancer Drug effects in C.elegans.) cell 169,442-456e4l 8.

[41]Searle, l.j., Meric, g., Porcelli, L, Sheppard, s.k., and Lucchini, S.S(2015) Variation of the distribution of siderophore biosynthetic genes across the environment and fecal populations of E.coli (Variation in siderophore biochemical gene distribution and production access environmental and facial publications of Escherichia coli.) public scientific library: combination 10, eOl 17906.

[41] Steiernage, T. (2006.) Maintenance of c.elegans (maintanence of c.elegans., (books of WormBook), 1-11. Tenalilon, O., Skumik, D., Picard, B, and Denamur, E. (2010.) Community genetics of commensal Escherichia coli (The marketing genetics of commercial Escherichia coli) Nature review: microbiology 8,207-.

[42] Tsang, W.Y. and Lemire, B.D (2003). Mitochondrial ATP synthase non-autonomously controls the developmental cells of C.elegans larvae (Mitochondrial ATP synthase control large degree development cell non-nanometronousbasis in Caenorhabditis elegans). Dev. kinetics (Dev Dyn) 226, 719-726.

[43] Vuong, h.e., Yano, j.m., Fung, t.c. and Hsiao, E.Y (2017), Microbiome and Host Behavior (The Microbiome and Host Behavior), annual review of neuroscience (Annu Rev Neurosci) 40,21-49 WHO (2002) iron deficiency anemia: assessment, prevention and control; programmer's guide (Iron specificity index, preservation, and control; a guide for program managers).

[44] Wiedemann, n. and Pfanner, n. (2017) Mitochondrial machinery for Protein Import and Assembly (mitochonddrial machinery for Protein Import and Assembly), annual review of biochemistry 86, 685-714.

[45] Wilson, b.r., Bogdan, a.r., Miyazawa, m., Hashimoto, k, and Tsuji, y. (2016.) siderophore in iron metabolism: from Mechanism to Therapy Potential (Sideroprese in Iron Metabolism: From Mechanism to Therapy Potential.) Trends in molecular medicine (Trends Mol Med) 22, 1077-.

[46] Xiao, x., Yeoh, b.s. and Vijay-Kumar, M. (2017). lipocalin 2: iron Homeostasis and Inflammation Emerging "players" (Lipocalin 2: An empirical Player in Iron Homeostasis and Inflammation), annual review of nutrition (Annu Rev Nutr) 37,103- "130.

[47] Yu, d.j., Hu, j., Huang, y, Shen, h.b., Qi, y, Tang, z.m., and Yang, J.Y. (2013). A template-free method for ATP binding site prediction using residual evolution image sparse representation and classifier integration (targetATPSite: a template-free method for ATP-binding sites prediction with a residual evolution image space prediction and classifier ensemble.) A journal of computational chemistry (J computer Chem) 34, 974-.

[48] Zhang, l., Ding, l., cheng, t.h., Dong, m.q., Chen, j., swell, a.k., Liu, x., Yates, j.r., 3rd, and Han, M. (2007), Systematic identification of c.elegans mircs proteins, miRNAs, and mRNA targets by their interaction with GW182 proteins AIN-l and AIN-2 (Systematic identification of c.elegans mircs proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-l and AIN-2), molecular Cell (Mol) 28, 598-.

[49] Zheng, t, Bullock, j.l. and Nolan, E.M, (2012) siderophore-mediated delivery of cargo to the cytoplasm of escherichia coli and pseudomonas aeruginosa: synthesis of monofunctional Enterobacteriaceae scaffolds and evaluation of the uptake of Enterobacteriaceae-cargo conjugates (Single-functionalized vector delivery to the cytoplasms of Escherichia coli and Pseudomonas aeruginosa: synthesis of monotransformed enterobacter scans and evaluation of enterobacter-cargo uptake). J.Chemicals J.J. (J.Chem Soc) 134, 18388-18400.

[50] Abergel, Rebecca j. et al enterobactin protonation and iron release: structural Characterization of the salicylic acid Coordination conversion in Iron enterobacterin (Enterobacter protocol and Iron Release: Structural Characterization of the salt Coordination Shift in Ferric Enterobacter.) Journal of the American Chemical Society 128.27(2006):8920-8931.PMC.Web.2018, 7.17.

[51] Anderson, G.J, (2018), Iron competition-Host counterattack (Iron Wars-The Host striks Back, new england medical journal, 379,2078, 2080.

[52] Arora NK and Verma M. (2017). Modified microplate method for rapid and efficient estimation of bacterially produced siderophore (3 Biotech.). 7: 381.

[53] Auerbach, m. and Macdougall, i. (2017). Increase in International hemodialysis (Hemodal Int) 21, S83-S92, History, efficacy, and toxicology (The available intravenous iron relations: History, efficacy, and toxicology).

[54] Brissot, p., Bardou-Jacquet, e., jouanole, a.m., and Loreal, o. (2011) iron disorders of genetic origin: the world of change (Iron disorders of genetic origin: a changing world) molecular medicine trends 17, 707-713.

[55] Cook, J.D. and Reddy, M.B. (1995) Efficacy of weekly iron supplementation compared to daily iron supplementation (Efficacy of weekly supplemented with daily iron supplementation.) European journal of clinical Nutr (Eur J Clin Nutr) 62, 117-.

[56] Goetz D.H., Holmes M.A., Borregard, N., Bluhm M.E., Raymond K.N., Strong R.K (2002) neutrophil lipoprotein NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition (The neutrophilic lipocalin NGAL is a bacteriostatic agent with siderophore-mediated iron acquisition) & molecular cell 2002.10: 1033-43.

[57] Jaeggi, T, Kortman, G.A., Moretti, D, Chassard, C, Holding, P, Dostal, A, Boekhorst, J, Timmerman, H.M., Swinkels, D.W., Tjalsma, H.et al (2015) Iron fortification adversely affects the Gut microbiome of Kenya infants, increases pathogen abundance, and induces enteritis (Iron diagnosis additive infections in Kenyan infections) 742.

[58] Kortman, g.a., Dutilh, b.e., Maathuis, a.j., Engelke, u.f., Boekhorst, j., Keegan, k.p., Nielsen, f.g., Betley, j., Weir, j.c., Kingsbury, z. et al (2015), in an in vitro model of TIM-2 of the human colon, the microbial metabolism shifts toward an adverse condition with supplemental iron [ microbiology frontier ] 6,1481.

[59] Koutroluubakis, I.E., Ramos-Rivers, C., Regueiro, M., Koutroumpakis, E., Click, B., Schoen, R.E., Hasshash, J.G., Schwartz, M., Swoger, J., Baidoo, L. et al (2015) Persistent or Recurrent Anemia Is Associated With Severe and Disabling Inflammatory Bowel Disease (Persistant or Recurrent Anima Associated With Cold liver and Disabling Inflammatory Bowel Disease). Clin gastroenterology and hepatology (Clin Gastroenterol liver 17613, 1760).

[60] Lund, E.K., Wharf, S.G., Fairweather-Tait, S.J., and Johnson, I.T (1999). Oral ferrous sulfate supplements increase the free radical generating capacity of the feces of healthy volunteers (Oral ferrous sulfate supplements) J.J.J.J.J.69, 250-255 J.J..

[61] Moretti, D., Goede, J.S., Zeder, C., Jeskra, M., Chatzinakou, V., Tjalsma, H., Melse-Boostra, A., Brittenham, G., Swinkels, D.W., and Zimmermann, M.B. (2015.) in young, iron-depleted women, Oral iron supplements increase iron modulators and reduce iron absorption from daily or twice-daily doses (Oral iron supplementation in diabetes mellitus and depletion in diabetes mellitus from either dry or bright-day doses in iron-depleted women Blood (Blood) 126,1981, 1989.

[62] Muckenthaler, m.u., rivela, s., hentz, m.w., and Galy, b. (2017.) red carpet for iron metabolism, cell 168, 344-361.

[63] Munoz, M., Gomez-Ramirez, S., and Garcia-Erce, J.A, (2009) Intravenous iron in inflammatory bowel disease (Intra virus in inflimator bowel disease), journal of World gastroenterology (World J Gastroenterol) 15, 4666-.

[64] Qi, B., Kniazeva, M. and Han, M. (2018.) Microbial Siderophore enterobacterin Promotes Mitochondrial Iron Uptake and Development in the Host by interacting with ATP Synthase (Microbial Siderophore Enterobacter microorganisms microorganism microorganisms ion Uptake and Development of the Host via Interaction with ATP Synthase.). cell 175,571-582 e5 ll.

[65] Riemer, J., Hoepken, H.H., Czerwinska, H., Robinson, S.R., and Dringen, R. (2004). Colorimetric phenazine-based assays for the quantification of iron in cultured cells, Analyzed biochemicals 331, 370-375.

[66] Sazawal, s., Black, r.e., Ramsan, m., Chwaya, h.m., Stoltzfus, r.j., Dutta, a., Dhingra, u., Kabole, i., Deb, s., Othman, m.k. et al (2006) effect of routine prophylactic supplementation of iron and folic acid on admission and mortality of preschool children in malaria highly-transmitting environments: lancet (Lancet) 367,133-143, a community-based randomized, procedural, administration to a host and clinical, and regulatory in preschool-level transmission setting.

[67] The cross-SNARE regulatory function of she, c, ratore, s.s., Yu, h, Gulbranson, d.r., Hua, r, Zhang, c., Schoppa, n.e., and she, j. (2015). Muncl8-l is essential for synaptic exocytosis (The trans-SNARE-regulating function of Muncl8-l is infectious to synthetic exocytosis): nature communication 6,8852.

[68] Stevens, g.a., Finucane, m.m., De-Regil, l.m., Paciorek, c.j., Flaxman, s.r., branch, f., Pena-rosa, j.p., Bhutta, z.a., Ezzati, m., and Nutrition Impact Model studies (Nutrition Impact Model Study), g. (2013), 1995-2011 children and pregnant women and non-pregnant women with global, regional, and national trends in hemoglobin concentrations and prevalence of global and severe anemia: analysis of systems with demographic data (Global, regional, and national transmissions in genetic control and prediction of total and segment and experience in childrens and prediction and non-prediction women for 1995. A systematic analysis of position-prediction data.) Lancet Global Health (Lancet Global Health) 1, el 6-25.

[69] Iron in Micronutrient powders Promotes undesirable Gut Microbiota in kenya Infants (Iron in Micronutrient Powder an ETnfavorable Gut Microbiota in Kenyan Infants) 9.

[70] WHO (2015), Global anemia Prevalence (The Global preference of anaemia).

[71] Yu, h., rathio, s.s., Shen, c., Liu, y., Ouyang, y., stowwell, m.h., and Shen, j. (2015). The important Role of Macromolecular Crowding (The scientific Role of Macromolecular crowing) is described in journal of The American society for chemistry 137, 12873-12883.

[72] Zhang, a.s. and ens, c.a. (2009) iron steady state: recently Identified Proteins Provide the idea of a Novel Control mechanism (Iron Homeostasis: centered Identified protein instruments in Novel Control Mechanisms), J.Biochem.J. (Journal of Biological Chemistry) 284, 711-715.

Sequence identification

SEQ ID NO.1

EntB

Amino acids

Escherichia coli

SEQ ID NO.2

EntD

Amino acids

Escherichia coli

SEQ ID NO.3

EntE

Amino acids

Escherichia coli

SEQ ID NO.4

EntE

Amino acids

Escherichia coli

SEQ ID NO.5

EntA

Amino acids

Escherichia coli

SEQ ID NO.6

EntC

Amino acids

Escherichia coli

Sequence listing

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Ile Pro Gln Asn Lys Val Asp Trp Ala Phe Glu Pro Gln Arg Ala Ala

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Leu Leu Ile His Asp Met Gln Asp Tyr Phe Val Ser Phe Trp Gly Glu

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Asn Cys Pro Met Met Glu Gln Val Ile Ala Asn Ile Ala Ala Leu Arg

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Lys Glu Gln Ser Asp Glu Asp Arg Ala Leu Leu Asn Asp Met Trp Gly

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Pro Gly Leu Thr Arg Ser Pro Glu Gln Gln Lys Val Val Asp Arg Leu

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Thr Pro Asp Ala Asp Asp Thr Val Leu Val Lys Trp Arg Tyr Ser Ala

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Phe His Arg Ser Pro Leu Glu Gln Met Leu Lys Glu Ser Gly Arg Asn

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Gln Leu Ile Ile Thr Gly Val Tyr Ala His Ile Gly Cys Met Thr Thr

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Ala Thr Asp Ala Phe Met Arg Asp Ile Lys Pro Phe Met Val Ala Asp

165 170 175

Ala Leu Ala Asp Phe Ser Arg Asp Glu His Leu Met Ser Leu Lys Tyr

180 185 190

Val Ala Gly Arg Ser Gly Arg Val Val Met Thr Glu Glu Leu Leu Pro

195 200 205

Ala Pro Val Pro Ala Ser Lys Ala Ala Leu Arg Glu Val Ile Leu Pro

210 215 220

Leu Leu Asp Glu Ser Asp Glu Pro Phe Asp Asp Asp Asn Leu Ile Asp

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Tyr Gly Leu Asp Ser Val Arg Met Met Ala Leu Ala Ala Arg Trp Arg

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Gln Ile Asp Phe Asp Pro Ala Thr Phe Gln Pro Glu Asp Leu Phe Trp

20 25 30

Leu Pro Tyr His Ala Ser Leu Thr Gly Trp Gly Arg Lys Arg Gln Ala

35 40 45

Glu His Leu Ala Gly Arg Ile Ala Ala Ala Tyr Ala Leu Arg Glu Val

50 55 60

Gly Glu Lys Arg Leu Pro Ala Ile Gly Asp Gln Arg Gln Pro Leu Trp

65 70 75 80

Pro Thr Pro Trp Phe Gly Ser Ile Ser His Cys Gly Gln Arg Ala Leu

85 90 95

Ala Val Ile Ala Asp Arg Pro Val Gly Val Asp Ile Glu Arg Arg Phe

100 105 110

Thr Pro Gln Leu Ala Ala Glu Leu Glu Ser Ser Ile Ile Ser Pro Ala

115 120 125

Glu Lys Thr Ala Leu Leu Arg Ser Gly Leu Pro Phe Pro Leu Ala Leu

130 135 140

Thr Leu Ala Phe Ser Ala Lys Glu Ser Gly Phe Lys Ala Cys His Pro

145 150 155 160

Asp Val Gln Ala Gly Val Gly Phe Asn Asp Phe Thr Leu Ala Ala Ile

165 170 175

Lys Glu Gly Asn Leu Arg Leu Arg Leu Ser Thr Val Glu Tyr Arg Leu

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Gln Trp Ile Gln Ala Gly Glu Tyr Ile Ile Thr Leu Cys Ala Pro

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Met Ser Ile Pro Phe Thr Arg Trp Pro Glu Glu Phe Ala Arg Arg Tyr

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Arg Glu Lys Gly Tyr Trp Gln Asp Leu Pro Leu Thr Asp Ile Leu Thr

20 25 30

Arg His Ala Ala Ser Asp Ser Ile Ala Val Ile Asp Gly Glu Arg Gln

35 40 45

Leu Ser Tyr Arg Glu Leu Asn Gln Ala Ala Asp Asn Leu Ala Cys Ser

50 55 60

Leu Arg Arg Gln Gly Ile Lys Pro Gly Glu Thr Ala Leu Val Gln Leu

65 70 75 80

Gly Asn Val Ala Glu Leu Tyr Ile Thr Phe Phe Ala Leu Leu Lys Leu

85 90 95

Gly Val Ala Pro Val Leu Ala Leu Phe Ser His Gln Arg Ser Glu Leu

100 105 110

Asn Ala Tyr Ala Ser Gln Ile Glu Pro Ala Leu Leu Ile Ala Asp Arg

115 120 125

Gln His Ala Leu Phe Ser Gly Asp Asp Phe Leu Asn Thr Phe Val Thr

130 135 140

Glu His Ser Ser Ile Arg Val Val Gln Leu His Asn Asp Ser Gly Glu

145 150 155 160

His Asn Leu Gln Asp Ala Ile Asn His Pro Ala Glu Asp Phe Thr Ala

165 170 175

Thr Pro Ser Pro Ala Asp Glu Val Ala Tyr Phe Gln Leu Ser Gly Gly

180 185 190

Thr Thr Gly Thr Pro Lys Leu Ile Pro Arg Thr His Asn Asp Tyr Tyr

195 200 205

Tyr Ser Val Arg Arg Ser Val Glu Ile Cys Gln Phe Thr Gln Gln Thr

210 215 220

Arg Tyr Leu Cys Ala Ile Pro Ala Ala His Asn Tyr Ala Met Ser Ser

225 230 235 240

Pro Gly Ser Leu Gly Val Phe Leu Ala Gly Gly Thr Val Val Leu Ala

245 250 255

Ala Asp Pro Ser Ala Thr Leu Cys Phe Pro Leu Ile Glu Lys His Gln

260 265 270

Val Asn Val Thr Ala Leu Val Pro Pro Ala Val Ser Leu Trp Leu Gln

275 280 285

Ala Leu Thr Glu Gly Glu Ser Arg Ala Gln Leu Ala Ser Leu Lys Leu

290 295 300

Leu Gln Val Gly Gly Ala Arg Leu Ser Ala Thr Leu Ala Ala Arg Ile

305 310 315 320

Pro Ala Glu Ile Gly Cys Gln Leu Gln Gln Val Phe Gly Met Ala Glu

325 330 335

Gly Leu Val Asn Tyr Thr Arg Leu Asp Asp Ser Ala Glu Lys Ile Ile

340 345 350

His Thr Gln Gly Tyr Pro Met Cys Pro Asp Asp Glu Val Trp Val Ala

355 360 365

Asp Ala Glu Gly Asn Pro Leu Pro Gln Gly Glu Val Gly Arg Leu Met

370 375 380

Thr Arg Gly Pro Tyr Thr Phe Arg Gly Tyr Tyr Lys Ser Pro Gln His

385 390 395 400

Asn Ala Ser Ala Phe Asp Ala Asn Gly Phe Tyr Cys Ser Gly Asp Leu

405 410 415

Ile Ser Ile Asp Pro Glu Gly Tyr Ile Thr Val Gln Gly Arg Glu Lys

420 425 430

Asp Gln Ile Asn Arg Gly Gly Glu Lys Ile Ala Ala Glu Glu Ile Glu

435 440 445

Asn Leu Leu Leu Arg His Pro Ala Val Ile Tyr Ala Ala Leu Val Ser

450 455 460

Met Glu Asp Glu Leu Met Gly Glu Lys Ser Cys Ala Tyr Leu Val Val

465 470 475 480

Lys Glu Pro Leu Arg Ala Val Gln Val Arg Arg Phe Leu Arg Glu Gln

485 490 495

Gly Ile Ala Glu Phe Lys Leu Pro Asp Arg Val Glu Cys Val Asp Ser

500 505 510

Leu Pro Leu Thr Ala Val Gly Lys Val Asp Lys Lys Gln Leu Arg Gln

515 520 525

Trp Leu Ala Ser Arg Ala Ser Ala

530 535

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Met Ala Ala Thr Met Arg Leu Thr Gly Arg Leu Gly His Cys Val Ser

1 5 10 15

Ala Ala Val Thr Gly Val Leu Pro Ala Val Ala Gly Ser Pro Leu Ala

20 25 30

Tyr Ser Asp Thr Asp Glu Phe Tyr Pro Val Ala Gly Gly Thr Met Ser

35 40 45

Gln His Leu Pro Leu Val Ala Ala Gln Pro Gly Ile Trp Met Ala Glu

50 55 60

Lys Leu Ser Glu Leu Pro Ser Ala Trp Ser Val Ala His Tyr Val Glu

65 70 75 80

Leu Thr Gly Glu Val Asp Ser Pro Leu Leu Ala Arg Ala Val Val Ala

85 90 95

Gly Leu Ala Gln Ala Asp Thr Leu Arg Met Arg Phe Thr Glu Asp Asn

100 105 110

Gly Glu Val Trp Gln Trp Val Asp Asp Ala Leu Thr Phe Glu Leu Pro

115 120 125

Glu Ile Ile Asp Leu Arg Thr Asn Ile Asp Pro His Gly Thr Ala Gln

130 135 140

Ala Leu Met Gln Ala Asp Leu Gln Gln Asp Leu Arg Val Asp Ser Gly

145 150 155 160

Lys Pro Leu Val Phe His Gln Leu Ile Gln Val Ala Asp Asn Arg Trp

165 170 175

Tyr Trp Tyr Gln Arg Tyr His His Leu Leu Val Asp Gly Phe Ser Phe

180 185 190

Pro Ala Ile Thr Arg Gln Ile Ala Asn Ile Tyr Cys Thr Trp Leu Arg

195 200 205

Gly Glu Pro Thr Pro Ala Ser Pro Phe Thr Pro Phe Ala Asp Val Val

210 215 220

Glu Glu Tyr Gln Gln Tyr Arg Glu Ser Glu Ala Trp Gln Arg Asp Ala

225 230 235 240

Ala Phe Trp Ala Glu Gln Arg Arg Gln Leu Pro Pro Pro Ala Ser Leu

245 250 255

Ser Pro Ala Pro Leu Pro Gly Arg Ser Ala Ser Ala Asp Ile Leu Arg

260 265 270

Leu Lys Leu Glu Phe Thr Asp Gly Glu Phe Arg Gln Leu Ala Thr Gln

275 280 285

Leu Ser Gly Val Gln Arg Thr Asp Leu Ala Leu Ala Leu Ala Ala Leu

290 295 300

Trp Leu Gly Arg Leu Cys Asn Arg Met Asp Tyr Ala Ala Gly Phe Ile

305 310 315 320

Phe Met Arg Arg Leu Gly Ser Ala Ala Leu Thr Ala Thr Gly Pro Val

325 330 335

Leu Asn Val Leu Pro Leu Gly Ile His Ile Ala Ala Gln Glu Thr Leu

340 345 350

Pro Glu Leu Ala Thr Arg Leu Ala Ala Gln Leu Lys Lys Met Arg Arg

355 360 365

His Gln Arg Tyr Asp Ala Glu Gln Ile Val Arg Asp Ser Gly Arg Ala

370 375 380

Ala Gly Asp Glu Pro Leu Phe Gly Pro Val Leu Asn Ile Lys Val Phe

385 390 395 400

Asp Tyr Gln Leu Asp Ile Pro Asp Val Gln Ala Gln Thr His Thr Leu

405 410 415

Ala Thr Gly Pro Val Asn Asp Leu Glu Leu Ala Leu Phe Pro Asp Val

420 425 430

His Gly Asp Leu Ser Ile Glu Ile Leu Ala Asn Lys Gln Arg Tyr Asp

435 440 445

Glu Pro Thr Leu Ile Gln His Ala Glu Arg Leu Lys Met Leu Ile Ala

450 455 460

Gln Phe Ala Ala Asp Pro Ala Leu Leu Cys Gly Asp Val Asp Ile Met

465 470 475 480

Leu Pro Gly Glu Tyr Ala Gln Leu Ala Gln Ile Asn Ala Thr Gln Val

485 490 495

Glu Ile Pro Glu Thr Thr Leu Ser Ala Leu Val Ala Glu Gln Ala Ala

500 505 510

Lys Thr Pro Asp Ala Pro Ala Leu Ala Asp Ala Arg Tyr Leu Phe Ser

515 520 525

Tyr Arg Glu Met Arg Glu Gln Val Val Ala Leu Ala Asn Leu Leu Arg

530 535 540

Glu Arg Gly Val Lys Pro Gly Asp Ser Val Ala Val Ala Leu Pro Arg

545 550 555 560

Ser Val Phe Leu Thr Leu Ala Leu His Ala Ile Val Glu Ala Gly Ala

565 570 575

Ala Trp Leu Pro Leu Asp Thr Gly Tyr Pro Asp Asp Arg Leu Lys Met

580 585 590

Met Leu Glu Asp Ala Arg Pro Ser Leu Leu Ile Thr Thr Asp Asp Gln

595 600 605

Leu Pro Arg Phe Ser Asp Val Pro Asn Leu Thr Ser Leu Cys Tyr Asn

610 615 620

Ala Pro Leu Thr Pro Gln Gly Ser Ala Pro Leu Gln Leu Ser Gln Pro

625 630 635 640

His His Thr Ala Tyr Ile Ile Phe Thr Ser Gly Ser Thr Gly Arg Pro

645 650 655

Lys Gly Val Met Val Gly Gln Thr Ala Ile Val Asn Arg Leu Leu Trp

660 665 670

Met Gln Asn His Tyr Pro Leu Thr Gly Glu Asp Val Val Ala Gln Lys

675 680 685

Thr Pro Cys Ser Phe Asp Val Ser Val Trp Glu Phe Phe Trp Pro Phe

690 695 700

Ile Ala Gly Ala Lys Leu Val Met Ala Glu Pro Glu Ala His Arg Asp

705 710 715 720

Pro Leu Ala Met Gln Gln Phe Phe Ala Glu Tyr Gly Val Thr Thr Thr

725 730 735

His Phe Val Pro Ser Met Leu Ala Ala Phe Val Ala Ser Leu Thr Pro

740 745 750

Gln Thr Ala Arg Gln Asn Cys Ala Thr Leu Lys Gln Val Phe Cys Ser

755 760 765

Gly Glu Ala Leu Pro Ala Asp Leu Cys Arg Glu Trp Gln Gln Leu Thr

770 775 780

Gly Ala Pro Leu His Asn Leu Tyr Gly Pro Thr Glu Ala Ala Val Asp

785 790 795 800

Val Ser Trp Tyr Pro Ala Phe Gly Glu Glu Leu Ala Gln Val Arg Gly

805 810 815

Ser Ser Val Pro Ile Gly Tyr Pro Val Trp Asn Thr Gly Leu Arg Ile

820 825 830

Leu Asp Ala Met Met His Pro Val Pro Pro Gly Val Ala Gly Asp Leu

835 840 845

Tyr Leu Thr Gly Ile Gln Leu Ala Gln Gly Tyr Leu Gly Arg Pro Asp

850 855 860

Leu Thr Ala Ser Arg Phe Ile Ala Asp Pro Phe Ala Pro Gly Glu Arg

865 870 875 880

Met Tyr Arg Thr Gly Asp Val Ala Arg Trp Leu Asp Asn Gly Ala Val

885 890 895

Glu Tyr Leu Gly Arg Ser Asp Asp Gln Leu Lys Ile Arg Gly Gln Arg

900 905 910

Ile Glu Leu Gly Glu Ile Asp Arg Val Met Gln Ala Leu Pro Asp Val

915 920 925

Glu Gln Ala Val Thr His Ala Cys Val Ile Asn Gln Ala Ala Ala Thr

930 935 940

Gly Gly Asp Ala Arg Gln Leu Val Gly Tyr Leu Val Ser Gln Ser Gly

945 950 955 960

Leu Pro Leu Asp Thr Ser Ala Leu Gln Ala Gln Leu Arg Glu Thr Leu

965 970 975

Pro Pro His Met Val Pro Val Val Leu Leu Gln Leu Pro Gln Leu Pro

980 985 990

Leu Ser Ala Asn Gly Lys Leu Asp Arg Lys Ala Leu Pro Leu Pro Glu

995 1000 1005

Leu Lys Thr Gln Ala Ser Gly Arg Ala Pro Lys Ala Gly Ser Glu

1010 1015 1020

Thr Ile Ile Ala Ala Ala Phe Ala Ser Leu Leu Gly Cys Asp Val

1025 1030 1035

Gln Asp Ala Asp Ala Asp Phe Phe Ala Leu Gly Gly His Ser Leu

1040 1045 1050

Leu Ala Met Lys Leu Ala Ala Gln Leu Ser Arg Gln Phe Ala Arg

1055 1060 1065

Gln Val Thr Pro Gly Gln Val Met Val Ala Ser Thr Val Ala Lys

1070 1075 1080

Leu Ala Thr Ile Ile Asp Gly Glu Glu Asp Ser Ser Arg Arg Met

1085 1090 1095

Gly Phe Glu Thr Ile Leu Pro Leu Arg Glu Gly Asn Gly Pro Thr

1100 1105 1110

Leu Phe Cys Phe His Pro Ala Ser Gly Phe Ala Trp Gln Phe Ser

1115 1120 1125

Val Leu Ser Arg Tyr Leu Asp Pro Gln Trp Ser Ile Ile Gly Ile

1130 1135 1140

Gln Ser Pro Arg Pro His Gly Pro Met Gln Thr Ala Thr Asn Leu

1145 1150 1155

Asp Glu Val Cys Glu Ala His Leu Ala Thr Leu Leu Glu Gln Gln

1160 1165 1170

Pro His Gly Pro Tyr Tyr Leu Leu Gly Tyr Ser Leu Gly Gly Thr

1175 1180 1185

Leu Ala Gln Gly Ile Ala Ala Arg Leu Arg Ala Arg Gly Glu Gln

1190 1195 1200

Val Ala Phe Leu Gly Leu Leu Asp Thr Trp Pro Pro Glu Thr Gln

1205 1210 1215

Asn Trp Gln Glu Lys Glu Ala Asn Gly Leu Asp Pro Glu Val Leu

1220 1225 1230

Ala Glu Ile Asn Arg Glu Arg Glu Ala Phe Leu Ala Ala Gln Gln

1235 1240 1245

Gly Ser Thr Ser Thr Glu Leu Phe Thr Thr Ile Glu Gly Asn Tyr

1250 1255 1260

Ala Asp Ala Val Arg Leu Leu Thr Thr Ala His Ser Val Pro Phe

1265 1270 1275

Asp Gly Lys Ala Thr Leu Phe Val Ala Glu Arg Thr Leu Gln Glu

1280 1285 1290

Gly Met Ser Pro Glu Arg Ala Trp Ser Pro Trp Ile Ala Glu Leu

1295 1300 1305

Asp Ile Tyr Arg Gln Asp Cys Ala His Val Asp Ile Ile Ser Pro

1310 1315 1320

Gly Ala Phe Val Lys Ile Gly Pro Ile Ile Arg Ala Thr Leu Asn

1325 1330 1335

Arg

<210> 5

<211> 248

<212> PRT

<213> Escherichia Coli (E. Coli)

<400> 5

Met Asp Phe Ser Gly Lys Asn Val Trp Val Thr Gly Ala Gly Lys Gly

1 5 10 15

Ile Gly Tyr Ala Thr Ala Leu Ala Phe Val Glu Ala Gly Ala Lys Val

20 25 30

Thr Gly Phe Asp Gln Ala Phe Thr Gln Glu Gln Tyr Pro Phe Ala Thr

35 40 45

Glu Val Met Asp Val Ala Asp Ala Gly Gln Val Ala Gln Val Cys Gln

50 55 60

Arg Leu Leu Ala Glu Thr Glu Arg Leu Asp Val Leu Ile Asn Ala Ala

65 70 75 80

Gly Ile Leu Arg Met Gly Ala Thr Asp Gln Leu Ser Lys Glu Asp Trp

85 90 95

Gln Gln Thr Phe Ala Val Asn Val Gly Gly Ala Phe Asn Leu Phe Gln

100 105 110

Gln Thr Met Asn Gln Phe Arg Arg Gln Arg Gly Gly Ala Ile Val Thr

115 120 125

Val Ala Ser Asp Ala Ala His Thr Pro Arg Ile Gly Met Ser Ala Tyr

130 135 140

Gly Ala Ser Lys Ala Ala Leu Lys Ser Leu Ala Leu Ser Val Gly Leu

145 150 155 160

Glu Leu Ala Gly Ser Gly Val Arg Cys Asn Val Val Ser Pro Gly Ser

165 170 175

Thr Asp Thr Asp Met Gln Arg Thr Leu Trp Val Ser Asp Asp Ala Glu

180 185 190

Glu Gln Arg Ile Arg Gly Phe Gly Glu Gln Phe Lys Leu Gly Ile Pro

195 200 205

Leu Gly Lys Ile Ala Arg Pro Gln Glu Ile Ala Asn Thr Ile Leu Phe

210 215 220

Leu Ala Ser Asp Leu Ala Ser His Ile Thr Leu Gln Asp Ile Val Val

225 230 235 240

Asp Gly Gly Ser Thr Leu Gly Ala

245

<210> 6

<211> 395

<212> PRT

<213> Escherichia Coli (E. Coli)

<400> 6

Met Glu Asp Asp Met Asp Thr Ser Leu Ala Glu Glu Val Gln Gln Thr

1 5 10 15

Met Ala Thr Leu Ala Pro Asn Arg Phe Phe Phe Met Ser Pro Tyr Arg

20 25 30

Ser Phe Thr Thr Ser Gly Cys Phe Ala Arg Phe Asp Glu Pro Ala Val

35 40 45

Asn Gly Asp Ser Pro Asp Ser Pro Phe Gln Gln Lys Leu Ala Ala Leu

50 55 60

Phe Ala Asp Ala Lys Ala Gln Gly Ile Lys Asn Pro Val Met Val Gly

65 70 75 80

Ala Ile Pro Phe Asp Pro Arg Gln Pro Ser Ser Leu Tyr Ile Pro Glu

85 90 95

Ser Trp Gln Ser Phe Ser Arg Gln Glu Lys Gln Thr Ser Ala Arg Arg

100 105 110

Phe Thr Arg Ser Gln Ser Leu Asn Val Val Glu Arg Gln Ala Ile Pro

115 120 125

Glu Gln Thr Thr Phe Glu Gln Met Val Ala Arg Ala Ala Ala Leu Thr

130 135 140

Ala Thr Pro Gln Val Asp Lys Val Val Leu Ser Arg Leu Ile Asp Ile

145 150 155 160

Thr Thr Asp Ala Ala Ile Asp Ser Gly Val Leu Leu Glu Arg Leu Ile

165 170 175

Ala Gln Asn Pro Val Ser Tyr Asn Phe His Val Pro Leu Ala Asp Gly

180 185 190

Gly Val Leu Leu Gly Ala Ser Pro Glu Leu Leu Leu Arg Lys Asp Gly

195 200 205

Glu Arg Phe Ser Ser Ile Pro Leu Ala Gly Ser Ala Arg Arg Gln Pro

210 215 220

Asp Glu Val Leu Asp Arg Glu Ala Gly Asn Arg Leu Leu Ala Ser Glu

225 230 235 240

Lys Asp Arg His Glu His Glu Leu Val Thr Gln Ala Met Lys Glu Val

245 250 255

Leu Arg Glu Arg Ser Ser Glu Leu His Val Pro Ser Ser Pro Gln Leu

260 265 270

Ile Thr Thr Pro Thr Leu Trp His Leu Ala Thr Pro Phe Glu Gly Lys

275 280 285

Ala Asn Ser Gln Glu Asn Ala Leu Thr Leu Ala Cys Leu Leu His Pro

290 295 300

Thr Pro Ala Leu Ser Gly Phe Pro His Gln Ala Ala Thr Gln Val Ile

305 310 315 320

Ala Glu Leu Glu Pro Phe Asp Arg Glu Leu Phe Gly Gly Ile Val Gly

325 330 335

Trp Cys Asp Ser Glu Gly Asn Gly Glu Trp Val Val Thr Ile Arg Cys

340 345 350

Ala Lys Leu Arg Glu Asn Gln Val Arg Leu Phe Ala Gly Ala Gly Ile

355 360 365

Val Pro Ala Ser Ser Pro Leu Gly Glu Trp Arg Glu Thr Gly Val Lys

370 375 380

Leu Ser Thr Met Leu Asn Val Phe Gly Leu His

385 390 395

Claims (37)

1. A method of treating iron deficiency in a subject in need thereof, comprising administering a therapeutically effective amount of enterobacterin (Ent) or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the therapeutically effective amount of Ent is isolated.

3. The method of claim 1, wherein said therapeutically effective amount of Ent comprises an analogue of Ent wherein said therapeutically effective amount of Ent.

4. The method of claim 3, wherein the Ent analog is selected from the group consisting of: TRENCAM, SERSAM, SER (3M) SAM, TRENSAM and TREN (3M) SAM.

5. The method of claim 1 or 4, wherein said Ent or said Ent analog is combined with a pharmaceutically acceptable carrier.

6. The method of claim 5, wherein the pharmaceutically acceptable carrier is a nutritional supplement.

7. The method of claim 1, wherein the iron deficiency comprises iron deficiency anemia.

8. The method of claims 1 and 7, wherein the subject in need thereof comprises a human subject.

9. A method of preventing iron deficiency in a subject in need thereof, comprising administering a prophylactically effective amount of enterobacterin (Ent), or a pharmaceutically acceptable salt thereof.

10. The method of claim 9, wherein the therapeutically effective amount of Ent is isolated.

11. The method of claim 9, wherein said therapeutically effective amount of Ent comprises an analogue of Ent wherein said therapeutically effective amount of Ent.

12. The method of claim 11, wherein the Ent analog is selected from the group consisting of: TRENCAM, SERSAM, SER (3M) SAM, TRENSAM and TREN (3M) SAM.

13. The method of claim 9 or 14, wherein said Ent or said Ent analog is combined with a pharmaceutically acceptable carrier.

14. The method of claim 13, wherein the pharmaceutically acceptable carrier is a nutritional supplement.

15. The method of claim 9, wherein the iron deficiency comprises iron deficiency anemia.

16. The method of claims 9 and 15, wherein the subject in need thereof comprises a human subject.

17. A therapeutic agent for treating iron deficiency in a subject, the therapeutic agent comprising an active ingredient represented by the following general formula (I):

and a pharmaceutically acceptable carrier.

18. The method of claim 17, wherein the therapeutically effective amount of the compound of formula I is isolated.

19. The method of claim 17, wherein the therapeutically effective amount of the compound of formula I comprises an analog of the compound of formula I wherein the therapeutically effective amount.

20. The method of claim 19, wherein the analog of the compound of formula I is selected from the group consisting of:

21. the method of claim 17 or 20, wherein the compound of formulae I-VI is combined with a pharmaceutically acceptable carrier.

22. The method of claim 21, wherein the pharmaceutically acceptable carrier is a nutritional supplement.

23. The method of claim 17, wherein the iron deficiency comprises iron deficiency anemia.

24. The method of claims 17 and 23, wherein the subject in need thereof comprises a human subject.

25. The method of claims 6,14 and 22, wherein the nutritional supplement comprises a probiotic.

26. A genetically modified probiotic for treating iron deficiency in a subject in need thereof, the genetically modified probiotic comprising a probiotic configured to express a heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of enterobacterin (Ent).

27. The genetically modified bacterium of claim 26, wherein the probiotic comprises an Enterobacter (Enterobacter) probiotic.

28. The genetically modified bacterium of claim 26, wherein the heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of Ent comprises a heterologous nucleotide operably linked to a promoter encoding one or more genes selected from the group consisting of: entA, entB, entC, entD, entE and entF.

29. The genetically modified bacterium of claim 26, wherein the heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of Ent comprises a heterologous nucleotide operably linked to a promoter encoding one or more of the amino acid sequences selected from the group consisting of: SEQ ID NO. 1-6.

30. The genetically modified bacterium of claim 26, wherein the subject in need thereof is a human subject.

31. The genetically modified bacterium of claim 30, wherein the iron deficiency comprises iron deficiency anemia.

32. A nutraceutical composition for treating iron deficiency in a subject in need thereof, comprising a probiotic configured to express a heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of enterobacterin (Ent) and an excipient.

33. The nutraceutical composition of claim 32, wherein the probiotic comprises an enterobacter probiotic.

34. The nutraceutical composition of claim 32, wherein the heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of Ent comprises a heterologous nucleotide operably linked to a promoter encoding one or more genes selected from the group consisting of: entA, entB, entC, entD, entE and entF.

35. The nutraceutical composition of claim 32, wherein the heterologous nucleotide operably linked to a promoter encoding one or more genes for the biosynthesis of Ent comprises a heterologous nucleotide operably linked to a promoter encoding one or more of the amino acid sequences selected from the group consisting of: SEQ ID NO. 1-6.

36. The nutraceutical composition of claim 32, wherein the subject in need thereof is a human subject.

37. The nutraceutical composition of claim 36, wherein the iron deficiency comprises iron deficiency anemia.

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